Nitrite formulations and their use as nitric oxide prodrugs

ABSTRACT

Compositions comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; and from about 4 weight parts to about 100 weight parts of a nitrite salt. Use of said composition in methods of reducing triglycerides or reducing C-reactive protein levels are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/842,550 filed on Apr. 7, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/150,155 filed on Oct. 2, 2018, which is acontinuation of U.S. patent application Ser. No. 14/827,100 filed onAug. 14, 2015, which is a continuation of U.S. patent application Ser.No. 14/610,492 filed on Jan. 30, 2015, now U.S. Pat. No. 9,119,823issued Sep. 1, 2015; which is a continuation of U.S. patent applicationSer. No. 13/668,776 filed on Nov. 5, 2012, now U.S. Pat. No. 8,962,038issued Feb. 24, 2015; which is a continuation of U.S. patent applicationSer. No. 12/856,957 filed on Aug. 16, 2010, now U.S. Pat. No. 8,303,995issued Nov. 6, 2012; which is a continuation-in-part of U.S. patentapplication Ser. No. 12/484,364 filed on Jun. 15, 2009, now U.S. Pat.No. 8,298,589 issued Oct. 30, 2012; which claims the benefit of priorityto U.S. Provisional Application No. 61/061,251 filed on Jun. 13, 2008,all of which are incorporated herein by reference in their entity.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to the field of cardiovascularhealth and performance. More particularly, several embodiments of theinvention relate to nitrite formulations and their use as nitric oxideprodrugs.

In one embodiment, the present invention is directed to a method ofreducing a patient's triglyceride level, comprising administering to thepatient a composition in a form of a lozenge dissolvable in the mouth,the composition comprising from about 40 weight parts to about 1000weight parts of a botanical nitrate source; from about 20 weight partsto about 500 weight parts of a botanical source of nitrite reductionactivity; from about 20 weight parts to about 500 weight partsL-citrulline; and from about 4 weight parts to about 100 weight parts ofa nitrite salt.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is directed to a composition,comprising from about 40 weight parts to about 1000 weight parts of abotanical nitrate source; from about 20 weight parts to about 500 weightparts of a botanical source of nitrite reduction activity; and fromabout 4 weight parts to about 100 weight parts of a nitrite salt.

In one embodiment, the present invention is directed to a method ofreducing a patient's triglyceride level, comprising administering to thepatient a composition in a form of a lozenge dissolvable in the mouth,the composition comprising from about 40 weight parts to about 1000weight parts of a botanical nitrate source; from about 20 weight partsto about 500 weight parts of a botanical source of nitrite reductionactivity; from about 20 weight parts to about 500 weight partsL-citrulline; and from about 4 weight parts to about 100 weight parts ofa nitrite salt.

In one embodiment, the present invention is directed to a method ofreducing a patient's C-reactive protein level, comprising administeringto the patient a composition in a form of a lozenge dissolvable in themouth, the composition comprising from about 40 weight parts to about1000 weight parts of a botanical nitrate source; from about 20 weightparts to about 500 weight parts of a botanical source of nitritereduction activity; from about 20 weight parts to about 500 weight partsL-citrulline; and from about 4 weight parts to about 100 weight parts ofa nitrite salt.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the present inventionaccording to several embodiments disclosed herein. The inventionaccording to several embodiments disclosed herein may be betterunderstood by reference to one or more of these figures in combinationwith the detailed description of specific embodiments presented herein.

FIG. 1. (top) Original tracing from blood and tissue aerobic andanaerobic nitrite reduction to NO. (bottom) Quantification of NOgeneration from nitrite under aerobic and anaerobic conditions in bloodand tissues after the addition of 200 μM nitrite. Data represent n=3-4for each tissue and NO quantified over 4 minutes. Inset:Compartment-specific fold increase from aerobic to anaerobic NOformation from nitrite.

FIGS. 2A-2F. Steady-state plasma and heart NOx and nitros(yl)ationlevels in mice following nitrite insufficiency and supplementation. Micewere fed a standard rodent chow or a low NOx chow±50 mg/L nitritesupplementation for 7 days at which time steady-state levels of plasmaand heart nitrite (FIG. 2A), nitrate (FIG. 2B), nitroso (FIG. 2C), andheart nitrosyl-heme (FIG. 2D) were measured. Mice fed a low NOx diet for7 days exhibited an exacerbated injury following myocardialischemia/reperfusion (I/R) (FIG. 2E) which was reversed in those animalssupplemented with nitrite. Representative immunoblots of eNOS, iNOS, andnNOS from myocardial tissue homogenates of mice on standard chow and lowNOx diet for 7 days reveal no changes in NOS protein expression, (FIG.2F). These data demonstrate that supplemental nitrite can protect theheart from damage following heart attack.

FIG. 3. Nitrite levels in heart tissue and plasma during 30 minutes ofischemia in a mouse model. These data demonstrate that nitrite isconsumed during ischemia and restored during reperfusion.

FIG. 4. Dietary nitrite insufficiency unmasks NO biochemistry (A)nitrite, (B) nitrate, (C) nitroso, and (D) NO-heme in eNOS knockout miceand is restored by supplementation in the drinking water. Methods: eNOSknockout mice on either standard diet or low NOx diet±50 mg/L nitritesupplementation were compared to C57 control mice on standard diet toreveal dietary nitrite can restore NO biochemistry in mice unable toproduce NO.

FIGS. 5A-5C. Mice were fed 50 mg/L nitrite supplement for 12 weeks,resulting in increased circulating nitrite and nitrate levels (FIGS. 5Aand 5B). The nitrite fed group had 20% less lesion formation on theabdominal aorta than the control group fed high fat diet with nitritefree water (FIG. 5C), demonstrating that nitrite supplementation caninhibit the progression of atherosclerosis in a mouse model ofatherosclerosis.

FIG. 6. Blood nitrite concentration after ingestion of an oralformulation containing sodium nitrite in a human model.

FIG. 7. Blood nitrate concentration after ingestion of an oralformulation containing L-arginine in a human model.

FIG. 8. Oxidation and reduction of nitrate, nitrite, and nitric oxide.

FIGS. 9A and 9B. Effect of a composition according to the presentinvention on NO concentration and blood NO levels compared to blood NOlevels from a nutritional supplement containing L-arginine andantioxidants.

FIGS. 10A-10D. (FIGS. 10A&B) A 30 day twice per day regimen of acomposition according to the present invention significantly increaseplasma levels of nitrite and nitrate to normal healthy levels; (FIG.10C) Patients taking a composition according to the present inventiondaily for 30 days saw a 10%-55% decrease in fasting triglycerides. (FIG.10D) Collectively all patients taking a composition according to thepresent invention daily experienced a statistically significantreduction in fasting triglycerides (data are ave±SEM of n=13 patients).

DETAILED DESCRIPTION OF THE INVENTION Significance

All life requires nitrogen-compounds. Nitrite (NO₂ ⁻) is such a compoundthat is naturally occurring in nature and biology. Over the years, thepharmacological stance on nitrite has undergone a surprisingmetamorphosis, from a vilified substance that generates carcinogenicnitrosamines in the stomach, to a life-saving drug that liberates aprotective agent (NO) during hypoxic events. Nitrite has beeninvestigated as a vasodilator in mammals for over 125 years and is aknown by-product of organic nitrate metabolism. There has been a recentre-discovery of some of the vasodilator actions of nitrite in physiologyalong with novel discoveries which may render nitrite a fundamentalmolecule in biology. Nitrite is emerging as an endogenous signalingmolecule and regulator of gene expression that can not only serve as adiagnostic marker but also as potential therapy of cardiovasculardisease. Up until recently nitrite was thought to be an inert oxidativebreakdown product of endogenous nitric oxide synthesis.

Certain embodiments of the present invention disclosed herein provide aformulation and a process to enhance and extend the therapeutichalf-life of nitrite and therefore increase nitric oxide (NO)bioavailability. Thus, several embodiments provide the basis for newpreventive or therapeutic strategies in diseases associated with NOinsufficiency and new guidelines for optimal health as well as extendthe therapeutic window in which one may intervene during a heart attack.Extension of nitrite half-life is desirable in the design ofcardioprotective therapeutics or preventative medicines. As such,several embodiments prevent the onset or progression of cardiovascularor heart disease and protect from myocardial infarction thrunitrite/nitrate supplementation. Furthermore, certain embodimentsprovide an extended half-life of nitrite, out to 1 hour, which is the“golden hour” in terms of recovery from heart attack and stroke. Thepossibility of modulating an endogenous signaling pathway (NO) known tobe involved in many physiological and pathophysiological events througha molecule found in certain foods is revolutionary and intriguing.

Nitrite, NO and Cardiovascular Disease

Ischemic heart disease, including myocardial infarction, remains theleading cause of morbidity and mortality in all industrialized nations(Myerburg, R. J. (2001). “Sudden cardiac death: exploring the limits ofour knowledge.” J Cardiovasc Electrophysiol 12(3): 369-81). There aretwo distinct components of damage to the heart in patients whoexperience acute myocardial infarction: ischemic injury and reperfusioninjury. The myocardium is able to tolerate brief periods of ischemia (anabsolute or relative shortage of the blood supply to an organ) asactivation of inherent, adaptive mechanisms can preserve energy levelsand prevent injury. These include switching metabolism to anaerobicglycolysis and fatty acid utilization, increasing glucose uptake, anddecreasing contractility. If ischemia persists however, the myocardiumwill develop a severe adenosine tri-phosphate (ATP) deficit, resultingin irreversible injury and culminating in cell death. Althoughreperfusion of ischemic tissues provides oxygen and metabolic substratesnecessary for the recovery and survival of reversibly injured cells,reperfusion itself paradoxically results in the acceleration of cellularnecrosis (Braunwald, E. and R. A. Kloner (1985). “Myocardialreperfusion: a double-edged sword?” J Clin Invest 76(5): 1713-9).Reperfusion is characterized by the formation of oxygen radicals uponreintroduction of molecular oxygen to ischemic tissues, resulting inwidespread lipid and protein oxidative modifications, mitochondrialinjury, as well as tissue apoptosis and necrosis (Nayler, W. G. (1981).“The role of calcium in the ischemic myocardium.” Am J Pathol 102(2):262-70; McCord, J. M., R. S. Roy, et al. (1985). “Free radicals andmyocardial ischemia. The role of xanthine oxidase.” Adv Myocardiol 5:183-9).

The loss of nitric oxide (NO) generation as a result of a dysfunctionalvascular endothelium is a very likely cause of heart disease (Esper, R.J., R. A. Nordaby, et al. (2006). “Endothelial dysfunction: acomprehensive appraisal.” Cardiovasc Diabetol 5: 4). Continuousgeneration of NO is essential for the integrity of the cardiovascularsystem and a decreased production and/or bioavailability of NO iscentral to the development of cardiovascular disorders (Ignarro, L. J.(2002). “Nitric oxide as a unique signaling molecule in the vascularsystem: a historical overview.” J Physiol Pharmacol 53(4 Pt 1): 503-14;Herman, A. G. and S. Moncada (2005). “Therapeutic potential of nitricoxide donors in the prevention and treatment of atherosclerosis.” EurHeart J 26(19): 1945-55). NO is a highly reactive and diffusible gasformed by three NO synthase (NOS) isoforms: neuronal NOS (nNOS),endothelial NOS (eNOS), and inducible NOS (iNOS). NO has beenextensively studied in the setting of ischemia-reperfusion(ischemia/reperfusion) injury. Previous studies clearly demonstrate thatthe deficiency of eNOS exacerbates myocardial ischemia/reperfusioninjury (Jones, S. P., W. G. Girod, et al. (1999). “Myocardialischemia-reperfusion injury is exacerbated in absence of endothelialcell nitric oxide synthase.” Am J Physiol 276(5 Pt 2): H1567-73; Sharp,B. R., S. P. Jones, et al. (2002). “Differential response to myocardialreperfusion injury in eNOS-deficient mice.” Am J Physiol Heart CircPhysiol 282(6): H2422-6), whereas the overexpression of eNOS (Jones, S.P., J. J. Greer, et al. (2004). “Endothelial nitric oxide synthaseoverexpression attenuates myocardial reperfusion injury.” Am J PhysiolHeart Circ Physiol 286(1): H276-82; Elrod, J. W., J. J. Greer, et al.(2006). “Cardiomyocyte-specific overexpression of NO synthase-3 protectsagainst myocardial ischemia-reperfusion injury.” Arterioscler ThrombVasc Biol 26(7): 1517-23), NO donor (Siegfried, M. R., C. Carey, et al.(1992). “Beneficial effects of SPM-5185, a cysteine-containing NO donorin myocardial ischemia-reperfusion.” Am J Physiol 263(3 Pt 2): H771-7;Pabla, R., A. J. Buda, et al. (1996). “Nitric oxide attenuatesneutrophil-mediated myocardial contractile dysfunction after ischemiaand reperfusion.” Circ Res 78(1): 65-72) or inhaled NO gas (Hataishi,R., A. C. Rodrigues, et al. (2006). “Inhaled nitric oxide decreasesinfarction size and improves left ventricular function in a murine modelof myocardial ischemia-reperfusion injury.” Am J Physiol Heart CircPhysiol 291(1): H379-84) therapy significantly protect the myocardium(Bolli, R. (2001). “Cardioprotective function of inducible nitric oxidesynthase and role of nitric oxide in myocardial ischemia andpreconditioning: an overview of a decade of research.” J Mol CellCardiol 33(11): 1897-918). NO possesses a number of physiologicalproperties that makes it a potent cardioprotective-signaling molecule.These include vasodilation and the inhibition of oxidative stress,platelet aggregation, leukocyte chemotaxis and apoptosis (Ignarro, L.J., G. M. Buga, et al. (1987). “Endothelium-derived relaxing factorproduced and release from artery and vein is nitric oxide.” Proc NatlAcad Sci U S A 84(24): 9265-9; X. L., A. S. Weyrich, et al. (1993).“Diminished basal nitric oxide release after myocardial ischemia andreperfusion promotes neutrophil adherence to coronary endothelium.” CircRes 72(2): 403-12; Li, J., C. A. Bombeck, et al. (1999). “Nitric oxidesuppresses apoptosis via interrupting caspase activation andmitochondrial dysfunction in cultured hepatocytes.” J Biol Chem 274(24):17325-33). NO synthesis is influenced by various cofactors such astetrahydrobiopterin, flavin mononucleotide and flavin adeninedinucleotide, the presence of reduced thiols, the endogenous NOSinhibitor asymmetric dimethylarginine (ADMA) and substrate and oxygenavailability. Without an adequate delivery of substrate and co-factors(conditions that exist during ischemia), NOS no longer produces NO butinstead transfers the free electrons to oxygen and thus produces freeoxygen radicals (Becker, B. F., C. Kupatt, et al. (2000). “Reactiveoxygen species and nitric oxide in myocardial ischemia and reperfusion.”Z Kardiol 89 Suppl 9: IX/88-91, hereby incorporated by referenceherein). Thus, there is a need for additional NO production in ischemictissues that may limit ischemia/reperfusion injury.

Nitrite is an oxidative breakdown product of NO that has been shown toserve as an acute marker of NO flux/formation (Kleinbongard, P., A.Dejam, et al. (2003). “Plasma nitrite reflects constitutive nitric oxidesynthase activity in mammals.” Free Radic Biol Med 35(7): 790-6).Nitrite has recently moved to the forefront of NO biology (Gladwin, M.T., A. N. Schechter, et al. (2005). “The emerging biology of the nitriteanion.” Nat Chem Biol 1(6): 308-14), as it represents a major storageform of NO in blood and tissues (Bryan, N. S. (2006). “Nitrite in nitricoxide biology: cause or consequence? A systems-based review.” Free RadicBiol Med 41(5): 691-701, hereby incorporated by reference herein). Inaddition to the oxidation of NO, nitrite is also derived from reductionof salivary nitrate by commensal bacteria in the mouth andgastrointestinal tract (Tannenbaum, S. R., A. J. Sinskey, et al. (1974).“Nitrite in human saliva. Its possible relationship to nitrosamineformation.” J Natl Cancer Inst 53: 79-84; van Maanen, J. M., A. A. vanGeel, et al. (1996). “Modulation of nitrate-nitrite conversion in theoral cavity.” Cancer Detect Prev 20(6): 590-6) as well as from dietarysources such as meat, vegetables and drinking water. Much of the recentfocus on nitrite physiology is due to its ability to be reduced to NOduring ischemic or hypoxic events (Zweier, J. L., P. Wang, et al.(1995). “Enzyme-independent formation of nitric oxide in biologicaltissues.” Nat Med 1(8): 804-9; Bryan, N. S., T. Rassaf, et al. (2004).“Cellular Targets and Mechanisms of Nitros(yl)ation: An Insight intoTheir Nature and Kinetics in vivo.” Proc. Natl. Acad Sci. USA 101(12):4308-4313, hereby incorporated by reference herein; Lundberg, J. O. andE. Weitzberg (2005). “NO generation from nitrite and its role invascular control.” Arterioscler Thromb Vasc Biol 25(5): 915-22; Bryan2006). Nitrite reductase activity in mammalian tissues has been linkedto the mitochondrial electron transport system (Walters, C. L., R. J.Casselden, et al. (1967). “Nitrite metabolism by skeletal musclemitochondria in relation to haem pigments.” Biochim Biophys Acta 143(2):310-8; Reutov, V. P. and E. G. Sorokina (1998). “NO-synthase andnitrite-reductase components of nitric oxide cycle.” Biochemistry (Mosc)63(7): 874-84; Kozlov, A. V., K. Staniek, et al. (1999). “Nitritereductase activity is a novel function of mammalian mitochondria.” FEBSLett 454(1-2): 127-30), protonation (Zweier, Wang et al. 1995),deoxyhemoglobin (Cosby, K., K. S. Partovi, et al. (2003). “Nitritereduction to nitric oxide by deoxyhemoglobin vasodilates the humancirculation.” Nature Medicine 9:1498-1505, hereby incorporated byreference herein), and xanthine oxidase (Alikulov, Z. A., P. L'Vov N, etal. (1980). “[Nitrate and nitrite reductase activity of milk xanthineoxidase]” Biokhimiia 45(9): 1714-8; Li, H., A. Samouilov, et al. (2004).“Characterization of the effects of oxygen on xanthine oxidase-mediatednitric oxide formation.”J Biol Chem 279(17): 16939-46; Webb, A., R.Bond, et al. (2004). “Reduction of nitrite to nitric oxide duringischemia protects against myocardial ischemia-reperfusion damage.” ProcNatl Acad Sci U S A 101(37): 13683-8). Nitrite can also transiently formnitrosothiols (RSNOs) under both normoxic and hypoxic conditions (Bryan,Rassaf et al. 2004) and a recent study by Bryan et al demonstrates thatsteady state concentrations of tissue nitrite and nitroso are affectedby changes in dietary NOx (nitrite and nitrate) intake (Bryan, N. S., B.O. Fernandez, et al. (2005). “Nitrite is a signaling molecule andregulator of gene expression in mammalian tissues.” Nat Chem Biol 1(5):290-7, hereby incorporated by reference herein). Previous studies haveshown that nitrite therapy prior to reperfusion protects against hepaticand myocardial ischemia/reperfusion injury (Webb, Bond et al. 2004;Duranski, M. R., J. J. Greer, et al. (2005). “Cytoprotective effects ofnitrite during in vivo ischemia-reperfusion of the heart and liver.” JClin Invest 115(5): 1232-1240). Additionally, experiments in primatesrevealed a beneficial effect of long-term application of nitrite oncerebral vasospasm (Pluta, R. M., A. Dejam, et al. (2005). “Nitriteinfusions to prevent delayed cerebral vasospasm in a primate model ofsubarachnoid hemorrhage.” Jama 293(12): 1477-84). Oral nitrite has alsobeen shown to reverse L-NAME induced hypertension and serve as analternate source of NO in vivo (Tsuchiya, K., Y. Kanematsu, et al.(2005). “Nitrite is an alternative source of NO in vivo.” Am J PhysiolHeart Circ Physiol 288(5): H2163-70).

A reduced NO availability is a hallmark of a number of cardiovasculardisorders. Hyperlipidemia, arterial hypertension, diabetes, smoking andaging are major risk factors for the manifestation of cardiovascularevents (Widlansky, M. E., N. Gokce, et al. (2003). “The clinicalimplications of endothelial dysfunction.” J. Am Coll Cardiol 42:1149-1160). Plasma nitrite reflects acute changes in endothelial NOSactivity in various mammals (Kleinbongard, Dejam et al. 2003) and thusmay provide an accurate measurement of patients at risk forcardiovascular events. A recent report by Kleinbongard et al.(Kleinbongard, P., A. Dejam, et al. (2006). “Plasma nitriteconcentrations reflect the degree of endothelial dysfunction in humans ”Free Radic Biol Med 40(2): 295-302), demonstrated that plasma nitritelevels progressively decrease with increasing cardiovascular risk load.Risk factors considered include age, hypertension, smoking, andhypercholesterolemia. Since nitrite acts as a protective molecule duringischemic events these data raise the intriguing possibility that theunderlying problem with these patients is their diminished nitritebioavailability. Since a substantial portion of steady state nitriteconcentrations in blood and tissue are derived from dietary sources(Bryan, Fernandez et al. 2005), modulation of nitrite and/or nitrateintake may provide a first line of defense for ischemic heart disease.

Therefore, several embodiments increase nitrite availability throughdiet or supplementation and provide an alternate route to increased NOavailability as well as conferring protection from ischemia/reperfusioninjury or other adverse cardiovascular event (e.g., heart attack,stroke, etc.). Since the half life of nitrite is on the order ofseconds, it can only be used acutely and repeatedly for any therapeuticbenefit. Dietary nitrite and nitrate supplementation for 7 days restoresNO homeostasis and protects the heart from ischemia/reperfusion injury(Bryan, N. S., J. W. Calvert, et al. (2007). “Dietary nitritesupplementation protects against myocardial ischemia-reperfusioninjury.” Proc Natl Acad Sci U S A, vol. 104 no. 48 19144-19149, herebyincorporated by reference herein) providing the first proof of conceptthat nitrite can be chronically administered and have a profound effecton outcome from heart attack.

Thus, several embodiments of the present invention provide a method forprolonging the half life of nitrate and extending therapeutic benefit.In one embodiment, the method comprises providing a nitrite salt,wherein the nitrite salt is provided in an amount ranging from about 10mg to about 100 mg; providing a nitrate salt, wherein the nitrate saltis provided in an amount ranging from about 50 mg to about 500 mg; andproviding an ascorbic acid, wherein the ascorbic acid is provided in anamount ranging from about 100 mg to about 2000 mg. The nitrate salt andascorbic acid are administered in combination with the nitrite salt. Thenitrite salt and the nitrate salt convert to nitrite in vivo. Theascorbic acid reduces the conversion of nitrite into N-nitroso compoundsin vivo, thereby extending the half-life of the nitrite. “Reduces” inthis context encompasses, but is not limited to, “minimizes” or“prevents.” The ascorbic acid is particularly advantageous in someembodiments because it reduces carcinogens (e.g., N-nitroso compounds)while increasing the bioavailability of nitrite. L-arginine is added insome embodiments. L-arginine, in some embodiments, serves as a substratefor nitric oxide synthase, thereby increasing NO formation, which inturn can increase nitrate and/or nitrite formation. In one embodiment,the nitrite salt, nitrate salt, ascorbic acid, and optionally L-arginineare provide to a mammal in a single dose.

Nitrite in NO Biology

Nitrite has recently been implicated in hypoxic vasodilation in thecirculation (Kim-Shapiro, D. B., M. T. Gladwin, et al. (2005). “Thereaction between nitrite and hemoglobin: the role of nitrite inhemoglobin-mediated hypoxic vasodilation.” Journal of InorganicBiochemistry 99: 237-246). As early as 1880, nitrite was described interms of its vasodilatory abilities (Reichert, E. T. and S. W. Mitchell(1880). “On the physiological action of potassium nitrite, with a noteon the physiological action on man.” Am J Med Sci 159: 158-180) and muchlater, Furchgott used acidified sodium nitrite to relax precontractedaortic strips in 1953 (Furchgott, R. F. and S. Bhadrakom (1953).“Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol,sodium nitrite and other drugs.” J Pharcol Exp Ther 108(2): 129-143).Both studies used supra-physiological concentrations of nitrite.However, recent studies have rediscovered the vasodilatory effect ofnitrite on forearm and systemic blood flow after nitrite infusion. Cosbyet al. (Cosby, Partovi et al. 2003) suggested that nitrite is a largeintravascular storage pool for NO and that nitrite bioactivation to NOcould dilate regions with tissue oxygen debt in the human circulation.However, earlier a study by Lauer et al. reported that nitrite lackedintrinsic vasodilatory properties (Lauer, T., M. Preik, et al. (2001).“Plasma nitrite rather than nitrate reflects regional endothelial nitricoxide synthase activity but lacks intrinsic vasodilator action.” ProcNatl Acad Sci USA 98(22): 12814-12819). This discrepancy is likely dueto kinetics and duration of infusion. Nitrite is found in high abundancethroughout the mammalian organ system (Bryan, Rassaf et al. 2004). It isnormally a short-lived, highly regulated ion in the circulation (200-600nM) with a half life in whole blood of 110 seconds (Kelm, M. (1999).“Nitric oxide metabolism and breakdown.” Biochim Biophys Acta 1411:273-289). Two independent groups have recently demonstrated thecytoprotective effects of nitrite in ischemia-reperfusion injury (Webb,Bond et al. 2004; Duranski, Greer et al. 2005). Duranski et al attributenitrite's protective effects to the reduction of nitrite to NO by thereductase activity of hemoglobin. The study by Webb et al using anisolated heart setup was in the absence of blood, clearly demonstratingthat the myocardial tissue itself can metabolize nitrite without theneed for hemoglobin. Moreover, inhalation of nitrite selectively dilatesthe pulmonary circulation under hypoxic conditions in vivo in sheep(Hunter, C. J., A. Dejam, et al. (2004). “Inhaled nebulized nitrite is ahypoxia-sensitive NO-dependent selective pulmonary vasodilator.” Nat Med10: 1122-1127). Experiments in primates revealed a beneficial effect oflong-term application of nitrite on cerebral vasospasm (Pluta, Dejam etal. 2005). Topical application of nitrite improves skin infections andulcerations (Hardwick, J. B., A. T. Tucker, et al. (2001). “A novelmethod for the delivery of nitric oxide therapy to the skin of humansubjects using a semi-permeable membrane.” Clin Sci (Lond) 100(4):395-400). Furthermore, in the stomach, nitrite-derived NO seems to playan important role in host defense (Duncan, C., H. Dougall, et al.(1995). “Chemical generation of nitric oxide in the mouth from theenterosalivary circulation of dietary nitrate.” Nat Med 1(6): 546-551;Dykhuizen, R. S., R. Frazer, et al. (1996). “Antimicrobial effect ofacidified nitrite on gut pathogens: importance of dietary nitrate inhost defense.” Antimicrob Agents Chemother 40(6): 1422-1425) and inregulation of gastric mucosal integrity (Bjorne, H. H., J. Petersson, etal. (2004). “Nitrite in saliva increases gastric mucosal blood flow andmucus thickness.” J Clin Invest 113(1): 106-114). All of these studiestogether along with the observation that nitrite can act as a marker ofNOS activity (Kleinbongard, Dejam et al. 2003) opened a new avenue forthe diagnostic and therapeutic application of nitrite, especially incardiovascular diseases, using nitrite as marker as well as an activeagent. However, it is still not known how and to what extent nitritereduction to NO occurs or how the NO-independent effects of nitritecontribute to the cytoprotection of ischemia/reperfusion insult.

The History of Nitrite and Nitrate

Nitrite has clearly emerged as an important molecule in biology, but itseffects on the endogenous NO pathway have been poorly investigated.Furthermore, its use as a potential therapy needs further safetyconsideration. Historically nitrite was considered a strong oxidant andpotential carcinogen. It has been in widespread use for many years. Itis used as a color fixative and preservation in meats and fish and isnaturally occurring in the soil and in vegetables. It is also used inmanufacturing diazo dyes, nitroso compounds, in the textile industry, inphotography and in the manufacture of rubber chemicals. Nitrite is alsoa common clinical and laboratory chemical that is used as a vasodilator(Reichert and Mitchell 1880), bronchodilator (Hunter, Dejam et al.2004), intestinal relaxant (Kozlov, A. V., B. Sobhian, et al. (2001).“Organ specific formation of nitrosyl complexes under intestinalischemia-reperfusion in rats involves NOS-independent mechanism(s).”Shock 15: 366-371) and used as an antidote for cyanide poisoning (Chen,K. K. and C. L. Rose (1952). “Nitrite and thiosulfate therapy in cyanidepoisoning.” J Am Med Assoc 149(2): 113-119). Considering its widespreaduse there have been many toxicological studies on acute and chronicexposure to nitrite. The fatal dose of nitrite is in the range of 22-23mg/kg body weight (from USFDA Generally Recognized as Safe FoodIngredient: Nitrates and Nitrites (Including Nitrosamines) 1972 byBattele-Columbus Laboratories and Department of Commence, SpringfieldVa.). Lower doses of either nitrite or nitrate have caused acutemethemoglobinemia, particularly in infants. In infants, a high nitriteor nitrate intake has been associated with “blue baby syndrome” causedby methemoglobinemia (Comly, H. H. (1945). “Cyanosis in infants causedby nitrates in well water.” JAMA 129: 112-116; Donohoe, W. E. (1949).“Cyanosis in infants with drinking water as a cause.” Paediatrics 3:308-311; Lecks, H. I. (1950). “Methemoglobinemia in infancy.” Am J DisChild 79: 117-123).

The major public health concern, particularly in the 1970s, was theendogenous formation of N-nitrosamines from nitrite and nitrate and itsrelevance to human cancer. The first report in the 1950s on thehepatocarcinogenic effects of N-nitrosodimethylamine (NDMA) (Magee, P.H. and J. M. Barnes (1956). “The production of malignant primary heptictumors in the rat by feeding dimethylnitrosamine ” Br. J. Cancer 10:114-122), and the suggestion that low molecular weight N-nitrosamines(RNNO) can be formed following nitrosation of various amines (Druckrey,H. and R. Preussmann (1962b). “Die Bilding carcinogener Nitrosamine amBeispiel des Tabakrauchs.” Naturewissenschaften 49: 498-499) ignited anenormous interest in N-nitrosamines and their association with cancer.Direct proof that such nitrosation reactions can occur was provided byEnder et al. (Ender, F., C. Havre, et al. (1964). “Isolation andidentification of a hepatotoxic factor in herring meat produced fromsodium nitrite preserved herring.” Naturwissenschaften 51: 637-638) whoidentified NDMA in nitrite preserved fish, and by Sander and Sief(Sander, J. and F. Seif (1969). “Bakterielle Reduction von nitrat immagen des menschen als ursache einer Nitrosamin-Bildung.”Arzneimittel-Forsch 19: 1091-1093) who demonstrated the in vivoformation of a nitrosamine in the acidic conditions of the humanstomach. Because of the potent carcinogenicity, wide environmentaloccurrence and ease of formation of nitrosamines, considerable efforthas been made to determine the levels of nitrite and nitrate in theexternal and internal human environment, and to assess exposure in orderto correlate it with human cancer at specific sites (Bartsch, H. and R.Montesano (1984). “Relevance of nitrosamines to human cancer.”Carcinogenesis 5(11): 1381-1393). Since the early 1980s there have beennumerous reports on the association of N-nitrosamines and human cancers(Craddock, V. M. (1983). “Nitrosamines and human cancer: proof of anassociation?” Nature 306: 638; Bartsch and Montesano 1984) but acausative link between nitrite exposure and cancer is still missing(Ward, M. H., T. M. deKok, et al. (2005). “Workgroup report:Drinking-water nitrate and health—recent findings and research needs.”Environ Health Perspect 113(11): 1607-14). Furthermore, a two year studyon the carcinogenicity of nitrite by NIH has conclusively found thatthere was no evidence of carcinogenic activity by sodium nitrite in maleor female rats or mice (Program, N. T. (2001). On The Toxicology andCarcinogenesis Studues of Sodium Nitrite. U. S. D. o. H. a. H. Services,National Institute of Health. NTP TR 495: 1-276). These negativeconnotations of nitrite and nitrate have led the United Statesgovernment to regulate and restrict the levels in food and drinkingwater. Early studies on nitrogen balance in humans and analyses of fecaland ileostomy samples indicated that nitrite and nitrate are formed denovo in the intestine. It was these early findings by Tannenbaum et al.(Tannenbaum, S. R., D. Fett, et al. (1978). “Nitrite and nitrate areformed by endogenous synthesis in the human intestine.” Science 200:1487-1488) that significantly altered conceptions of human exposure toexogenous nitrite and nitrates and represented the original observationsthat would eventually lead to the discovery of the L-arginine: NOpathway. Prior to these studies it was thought that steady-state levelsof nitrite and nitrate originated solely from the diet and fromnitrogen-fixing enteric bacteria. Endogenous sources of nitrite inmammals are derived from: 1. oxidation of endogenous nitric oxide, 2.nutritional sources such as meat, vegetable and drinking water, 3.reduction of salivary nitrate by commensal bacteria in the mouth andgastrointestinal tract. The discovery of the NO pathway and the emergingbiomedical applications of nitrite and nitrate necessitate a paradigmshift on the role of nitrite and nitrate in physiology.

Nitrate/Nitrite Reduction to NO

Humans, unlike prokaryotes, are thought to lack the enzymatic machineryto reduce nitrate back to nitrite. However, due to the commensalbacteria that reside within the human body it has been demonstrated thatthese bacteria can reduce nitrate thereby supplying an alternativesource of nitrite (Goaz, P. W. and H. A. Biswell (1961). “Nitritereduction in whole saliva.” J Dent Res 40: 355-365; Tannenbaum, Sinskeyet al. 1974; Ishiwata, H., A Tanimura, et al. (1975). “Nitrite andnitrate concentrations in human saliva collected from salivary ducts.” JFood Hyg Soc Jpn 16: 89-92; van Maanen, van Geel et al. 1996). Thereforedietary and enzymatic sources of nitrate are now a potentially largesource of nitrite in the human body. Nitrate is rapidly absorbed in thesmall intestines and readily distributed throughout the body (Walker, R.(1996). “The metabolism of dietary nitrites and nitrates.” Biochem SocTrans 24(3): 780-785). As much as 25% of the ingested nitrate isactively taken up by the salivary glands to be excreted in the saliva(Spiegelhalder, B., G. Eisenbrand, et al. (1976). “Influence of dietarynitrate on nitrite content of human saliva: possible relevance to invivo formation of N-nitroso compounds.” Food Cosmet Toxicol 14:545-548). Approximately 20% of the salivary nitrate is then reduced tonitrite by bacteria in the mouth (Spiegelhalder, Eisenbrand et al. 1976)and then disproportionates with formation of NO after entering theacidic environment of the stomach. This nitrate pathway to NO has beenshown to help reduce gastrointestinal tract infection, increase mucousbarrier thickness and gastric blood flow (Pique, J. M., B. J. Whittle,et al. (1989). “The vasodilator role of endogenous nitric oxide in therat gastric microcirculation.” Eur. J. Pharmacol 174(2-3): 293-296;Brown, J. F., P. J. Hanson, et al. (1992). “Nitric oxide donors increasemucus gel thickness in rat stomach.” Eur. J. Pharmacol 223(1): 103-104;McKnight, G. M., L. M. Smith, et al. (1994). “Chemical synthesis ofnitric oxide in the stomach from dietary nitrate in humans.” Gut 40(2):211-214; Walker 1996). The concentrations of nitrate in drinking waterare usually <10 mg/L in the absence of bacterial contamination (Kross,B. C., G. R. Hallberg, et al. (1993). “The nitrate concentration ofprivate well water in Iowa.” Am J Public Health 83(2): 270-272).Vegetables, especially beets, celery, and leafy vegetables like lettuceand spinach are rich in nitrates (Meah, M. N., N. Harrison, et al.(1994). “Nitrate and nitrite in foods and the diet.” Food Addit Contam11(4): 519-532; Walker 1996; Vallance, P. (1997). “Dietary nitrate:poison or panacea?” Gut 40(2): 211-214). Other vegetables containnitrate at lower concentrations, but because they are consumed ingreater quantity, they may contribute more nitrate and thus nitrite fromthe diet. For the average population, most nitrate exposure (86%) comesfrom vegetables, whereas the primary contributors to nitrite intake arecured meats (39%), baked goods and cereals (34%), and vegetables (16%).The National Research Council report The Health Effects of Nitrate,Nitrite, and N-Nitroso Compounds (NRC 1981) reported estimates ofnitrite and nitrate intake based on food consumption tables. They reportthat the average total nitrite and nitrate intake in the U.S. was 0.77mg and 76 mg, respectively per day. Nitrite and nitrate are excreted inthe kidneys. Nitrate is excreted in the urine as such or afterconversion to urea (Green, L. C., K. Ruiz de Luzuriaga, et al. (1981).“Nitrate biosynthesis in man ” Proc. Natl. Acad Sci. USA 78(12):7764-7768). Clearance of nitrate from blood to urine approximates 20ml/min in adults (Wennmalm, A., G. Benthin, et al. (1993). “Metabolismand excretion of nitric oxide in humans. An experimental and clinicalstudy.” Circ Res 73(6): 1121-1127), indicating considerable renaltubular reabsorption of this ion. There is little detectable nitrite ornitrate in feces (Bednar, C. and C. Kies (1994). “Nitrate and Vitamin Cfrom fruits and vegetables: impact of intake variations on nitrate andnitrite excretions in humans.” Plant Foods Hum Nutr 45(1): 71-80). Thereis some loss of nitrate and nitrite in sweat, but is not a major routeof excretion (Weller, R., S. Pattullo, et al. (1996). “Nitric oxide isgenerated on the skin surface by reduction of sweat nitrate.” J InvestDermatol 107(3): 327-331). Assuming the human body (70 kg) produces 1.68mmole NO per day (based on 1 μmole/kg/hr NO production), an averagedaily intake of 0.77 mg of nitrite would equate to 11.1 μmoles per dayand 76 mg nitrate would equate to 894 μmoles per day or roughly 1 mmoleNOx per day from diet. This almost matches what the human body makesfrom NO, assuming most of the NO goes to stepwise oxidation to nitriteand nitrate.

Nitrite Physiology

The endogenous production of NO by NOS has been established as playingan important role in vascular homeostasis, neurotransmission, and hostdefense mechanisms (Moncada, S., R. M. J. Palmer, et al. (1991). “Nitricoxide: physiology, pathophysiology and pharmacology.” Pharmacol Rev43(2): 109-142). The major pathway for NO metabolism is the stepwiseoxidation to nitrite and nitrate (Yoshida, K., K. Kasama, et al. (1983).“Biotransformation of nitric oxide, nitrite and nitrate.” Int Arch OccupEnviron Health 52: 103-115). In plasma or other physiological fluids orbuffers, NO is oxidized almost completely to nitrite, where it remainsstable for several hours (Kelm, M., M. Feelisch, et al. (1992). TheBiology of nitric oxide. Physiological and Clinical Aspects. S. Moncada,M. A. Marietta, J. B. Hibbs and E. A. Higgs. London, Portland Press. 1:319-322, hereby incorporated by reference herein; Grube, R., M. Kelm, etal. (1994). The Biology of Nitric Oxide. Enzymology, Biochemistry, andImmunology. S. Moncada, M. Feelisch, R. Busse and E. A. Higgs. London,Portland Press. 4: 201-204, hereby incorporated by reference herein);however, the half life of NO2— in human whole blood is about 110 seconds(Kelm 1999).

The oxidation of NO by molecular oxygen is second order with respect toNO:

2NO+O2→2NO₂  (1)

2NO+2NO₂→2N₂O₃→  (2)

2N₂O₃+2H₂O→4NO₂ ⁻+4H⁺→  (3)

whereby NO₂, N₂O₃ and NO₂ ⁻ represent nitrogen dioxide, dinitrogentrioxide and nitrite, respectively. It should be noted that N2O3 is apotent nitrosating agent by virtue of its ability to generate thenitrosonium ion (NO⁺). NO and nitrite are rapidly oxidized to nitrate inwhole blood. As stated above, the half life of NO₂ ⁻ in human blood isabout 110 seconds (Kelm 1999). Nitrate on the other hand has acirculating half life of 5-8 hours (Tannenbaum, S. R. (1994). “Nitrateand nitrite: origin in humans.” Science 205: 1333-1335, herebyincorporated by reference herein; Kelm, M. and K. Yoshida (1996).Metabolic Fate of Nitric Oxide and Related N-oxides. Methods in NitricOxide Research. M. and J. S. Stamler. Chichester, John Wiley and Sons:47-58, hereby incorporated by reference herein). Although the mechanismsby which NO and NO₂ ⁻ are converted to NO₃ ⁻ in vivo are not entirelyclear, there are several possibilities. During fasting conditions withlow intake of nitrite/nitrate, enzymatic NO formation from NOS accountsfor the majority of nitrite (Rhodes, P., A. M. Leone, et al. (1995).“The L-arginine:nitric oxide pathway is the major source of plasmanitrite in fasted humans.” Biochem Biophys Res Commun 209: 590-596).

NO production from nitrite has been described in infarcted heart tissue(Zweier, J. L., et al., Enzyme-independent formation of nitric oxide inbiological tissues. Nature Medicine, 1995. 1(8): p. 804-809). Nitritereductase activity in mammalian tissues has been linked to themitochondrial electron transport system (Walters, C. L., R. J.Casselden, and A. M. Taylor, Nitrite metabolism by skeletal musclemitochondria in relation to haem pigments. Biochim Biophys Acta, 1967.143: p. 310-318; Reutov, V. P. and E. G. Sorokina, NO-synthase andnitrite-reductase components of nitric oxide cycle. Biochemistry (Mosc),1998. 63(7): p. 874-884; Kozlov, A. V., K. Staniek, and H. Nohl, Nitritereductase activity is a novel function of mammalian mitochondria. FEBSLett, 1999. 454: p. 127-130; Nohl, H., et al., Mitochondria recyclenitrite back to the bioregulator nitric monoxide. Acta Biochim Pol,2000. 47: p. 913-921; Tischner, R., E. Planchet, and W. M. Kaiser,Mitochondrial electron transport as a source for nitric oxide in theunicellular green algae Chlorella sorokiniana. FEBS Lett, 2004. 576: p.151-155), protonation (Zweier, J. L., et al., Enzyme-independentformation of nitric oxide in biological tissues. Nature Medicine, 1995.1(8): p. 804-809; Hunter, C. J., et al., Inhaled nebulized nitrite is ahypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat Med,2004. 10: p. 1122-1127), deoxyhemoglobin (Hunter, C. J., et al., Inhalednebulized nitrite is a hypoxia-sensitive NO-dependent selectivepulmonary vasodilator. Nat Med, 2004. 10: p. 1122-1127; Cosby, K., etal., Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilatesthe human circulation. Nature Medicine, 2003. 9: p. 1498-1505), andxanthine oxidase (Li, H., et al., Characterization of the effects ofoxygen on xanthine oxidase-mediated nitric oxide formation. J. BiolChem, 2004. 279: p. 16939-16946; Alikulov, Z. A., N. P. L'vov, and V. L.Kretovich, Nitrate and nitrite reductase activity of milk xanthineoxidase. Biokhimiia, 1980. 45(9): p. 1714-1718; Webb, A., et al.,Reduction of nitrite to nitric oxide during ischemia protects againstmyocardial ischemia-reperfusion damage. Proc Natl Acad Sci USA, 2004.101(13683-13688)). Mitochondrial nitrite reduction has been shown tooccur by ubiquinol (Kozlov, A. V., K. Staniek, and H. Nohl, Nitritereductase activity is a novel function of mammalian mitochondria. FEBSLett, 1999. 454: p. 127-130; Nohl, H., et al., The multiple functions ofcoenzyme Q. Bioorg Chem, 2001. 29(1): p. 1-13) and cytochrome c oxidase(Castello, P. R., et al., Mitochondrial cytochrome oxidase producesnitric oxide under hypoxic conditions: implications for oxygen sensingand hypoxic signaling in eukaryotes. Cell Metab, 2006. 3(4): p. 277-87)with subsequent binding of the NO produced to cytochrome bc1 site ofcomplex III or complex IV resulting in oxygen-dependent reversibleinhibition of mitochondrial respiration (Takehara, Y., et al.,Oxygen-dependent reversible inhibition of mitochondrial respiration bynitric oxide. Cell Struct Funct, 1996. 21(4): p. 251-8). The acidicreduction of nitrite requires protonation and a one-electron reduction.The relatively low pKa of nitrite (3.34) (Principles of ModernChemistry. Third ed, ed. D. W. Oxtoby and N. H. Nachtrieb. 1996, FortWorth: Harcourt Brace College Publishers. 848) limits this activity inphysiology but it can occur in the stomach or during ischemic eventswhen tissue pH falls. Since many different pathways have been shown tobe able to reduce nitrite but require different conditions andsubstrates for optimal nitrite reduction, it is likely that all pathwaysmay become relevant but at different oxygen tension, substrateavailability, and perhaps even compartment specific needs.

The evolution of nitrite from a vilified substance that generatescarcinogenic nitrosamines in the stomach, to a life-saving drug thatliberates a protective agent (NO) during hypoxic events, as well asperforms many actions independent of NO, warrants a re-evaluation ofnitrite in biology. With nitrite acting as both an end product of NOsynthesis and a reservoir for NO, it is therefore a critical homeostaticmolecule in NO biology.

The collective evidence reviewed in this section strongly supports thenotion that there is a fundamental and physiological basis fordeveloping nitrite-based therapeutics. It is not understood how orallyingested nitrite (pKa 3.8) can survive the acidic environment of thestomach (pH 1-2). Furthermore, once nitrite is absorbed into thebloodstream it is known to be quickly oxidized to nitrate with a halflife of 110 seconds. Surprisingly, several embodiments of the presentinvention demonstrate that orally administered nitrite in specificcombination with nitrate and ascorbic acid can extend the therapeuticrange of nitrite from seconds to tens of minutes providing a novelapproach to treat or reduce injury from heart attack with nitrite. Insome embodiments ascorbic acid reduces endogenous nitrosation reactionin the gastrointestinal tract, which enhances the half life of nitrite.In some embodiments, the nitrate provides an additional source ofnitrite, again extending the functional half life, e.g., by increasingstores, of nitrite.

The human diet exerts important long-term effects on vital bodyfunctions and thereby makes an important contribution to health anddisease. While high intake of cholesterol, saturated fat, salt, andsugar are associated with a greater risk for cardiovascular disease,conventional wisdom has it that the opposite is true for abundantconsumption of fruits and vegetables. A diet rich in fruits andvegetables is associated with a lower risk of certain forms of cancerand cardiovascular disease. Recent epidemiological studies suggest acardioprotective action afforded specifically by green leafy vegetables.Green leafy vegetables such as spinach and lettuce, in addition to beingrich in antioxidants are especially rich in nitrite and nitrate as areberries, grapes, and a few other fruits. The high content of nitrite andnitrate is a major factor contributing to the positive health effects ofcertain vegetables via bioconversion to NO which exerts protectiveeffects on the cardiovascular system. A continuous intake of nitrite-and nitrate-containing food such as green leafy vegetables and berriesmay ensure that blood and tissue levels of NO are maintained at a levelsufficient to compensate for any disturbances in endogenous NOsynthesis. Dietary source of NO metabolites could therefore improvecirculation and oxygen delivery and lead to better health and increasedenergy. This dietary pathway may therefore not only provide essentialnutrients for NO production but also provide a rescue pathway for peopleat risk for cardiovascular disease. Several embodiments provide thenutrition and protection of a high vegetable diet in the form of a dailysupplement formulation which renders subjects protected from injury fromheart attack or other cardiovascular events, i.e. stroke, pulmonaryembolism. This strategy including nitrite/nitrate supplementation incombination with ascorbic acid may serve as an inexpensivecardioprotective regimen which may delay or reduce the onset orprogression of cardiovascular or heart disease and protect frommyocardial infarction.

Several embodiments of the invention are particularly advantageousbecause they provide a supplement formulation of nitrite, nitrate andVitamin C. Although, in some embodiments, such amounts may be found in ahigh vegetable diet, the time it would take to consume the requiredassortment of vegetables as well as the impact on the digestive systemwould adversely impact the absorption and/or bioavailability of thenitrite, nitrate and Vitamin C Moreover, the reaction of other compoundsand nutrients in the naturally occurring vegetable assortment may alsoadversely impact the impact the absorption and/or bioavailability of thenitrite, nitrate and Vitamin C Thus, a supplement of nitrite, nitrateand Vitamin C in e.g., daily dose formulations are advantageous inseveral embodiments because it increases the absorption and/orbioavailability of the formulation. In some embodiments, the formulationcomprises purified or isolated nitrite, nitrate and Vitamin C. In otherembodiments, the formulation consists essentially of purified orisolated nitrite, nitrate and Vitamin C. In yet other embodiments, theformulation consists of purified or isolated nitrite, nitrate andVitamin C

In other embodiments, the formulation consists essentially of purifiedor isolated nitrite, nitrate, Vitamin C, and L-arginine. In yet otherembodiments, the formulation consists of purified or isolated nitrite,nitrate, Vitamin C, and L-arginine.

In one embodiment, “consists essentially of” means the composition mayfurther contain one or more components selected from the groupconsisting of water and flavorants.

With 1 in every 3 men and 1 in every 10 women in the U.S. expected todevelop some major cardiovascular disease before reaching age 60 is itdesirable to take preventive measures now to enhance cardiovascularhealth.

In one embodiment, the present invention can provide a novel therapy forpatients experiencing myocardial infarction or stroke. Nitrite has beenshown to be protective in animal models of stroke and both cardiac andhepatic ischemia-reperfusion injury. Conversely, nitrite insufficiencyis associated with increased injury from ischemia-reperfusion insult.However, because of the short half-life of nitrite in the circulation(110 sec), the therapeutic window for nitrite alone is very narrow.Therefore, several embodiments provide patients with an extended-releaseformulation comprising nitrite, among additional components, to be usedupon onset of symptoms to provide at least some protection from injuryuntil the patient can be provided with reperfusion therapy, such as in ahospital setting.

In several embodiments, the present invention relates to the use ofsupplemental nitrite in combination with nitrate and vitamin C (ascorbicacid) as a preventive agent in cardiovascular disease. In someembodiments nitrate acts as an extended release nitrite source that isabsorbed and re-circulated through the enterosalivary pathway and isreduced to nitrite by commensal bacteria in the mouth. In someembodiments nitrite acts as a reservoir for nitric oxide activity.Reduced nitric oxide availability is a hallmark of a number ofcardiovascular disorders and plasma nitrite levels progressivelydecrease with increasing cardiovascular risk load. Therefore, severalembodiments provide a sufficient daily intake of nitrite, which isbeneficial to optimal cardiovascular health. A typical Western diet islow in nitrite and nitrate compared to a vegetarian or Mediterraneandiet and may therefore account for the increased incidence ofcardiovascular disease in the United States, Europe, and other developedcountries. A daily nitrite supplementation may provide the missingnutrient, analogously to a daily multivitamin The Nobel Prize inPhysiology or Medicine was awarded in 1998 for the discovery of nitricoxide in the cardiovascular system. Maintaining nitric oxideavailability is essential for optimal health, particularly for those atrisk for cardiovascular events, and therefore, in several embodiments,supplemental nitrite acts to increase the reservoir of nitric oxidewhich can be bio-activated upon need as a prevention rather than atreatment or therapy once disease has occurred.

In one embodiment, the present invention relates to a formulation for analternate source of nitric oxide during cardiovascular exercise and/ormuscle training. In a further embodiment, the formulation furthercomprises L-arginine. L-arginine is a natural amino acid substrate fornitric oxide synthase enzymes which produces L-citrulline and NO fromL-arginine in a complex reaction requiring oxygen. L-arginine can begiven as a pre-workout drink to saturate the NOS enzyme to producesufficient NO and dilate vessels. However, under conditions wheremuscles are working during anaerobic metabolism, oxygen availability isdiminished and therefore NOS can no longer produce NO. Therefore analternate substrate must be supplied to produce NO under anaerobicconditions. The substrate then becomes nitrite. Several embodimentssupply blood and muscles with nitrite before a workout, which providesan additional source of NO during the workout and improves muscle bloodflow during exercise, thereby enhancing performance and muscle buildingcapacity. In one embodiment, sodium nitrite is added to existing workoutbeverage formulations, thereby increasing NO and providing sufficient NObefore during and/or after a workout. Since the L-aginine:NO pathway isnot functional during workout, the addition of nitrite provides thesubstrate for anaerobic formation of NO, an alternate pathway for NOgeneration. So instead of increasing NO production before and after aworkout through the L-arginine:NO pathway, the presence of nitrite incertain embodiments of the formulation will allow NO production fromnitrite reduction during the workout, a time at which it is advantageousto increase blood flow and supply the muscles with essential nutrientsand oxygen.

In one embodiment, the present invention relates to a compositioncomprising a nitrite salt, a nitrate salt, and ascorbic acid.

Any positively-charged ion safe for use as a food additive or acomponent of a pharmaceutical formulation can be used as the counterionto nitrite in the nitrite salt or the counterion to nitrate in thenitrate salt. In one embodiment, the positively-charged ion is aninorganic ion. In a further embodiment, the positively-charged ion isselected from the group consisting of sodium and potassium; e.g., thenitrite salt is sodium nitrite or potassium nitrite and the nitrate saltis sodium nitrate or potassium nitrate.

Any proportions of the components of the composition can be used. In oneembodiment, the composition comprises from about 1 weight part to about8 weight parts sodium nitrite, from about 5 weight parts to about 50weight parts sodium nitrate, and from about 20 weight parts to about 200weight parts ascorbic acid.

In one embodiment, the composition further comprises L-arginine. In afurther embodiment, the composition comprises from about 20 weight partsto about 200 weight parts L-arginine.

In some embodiments, sodium nitrite is included in a range of about 0.01mg/kg to about 15 mg/kg. In some embodiments, sodium nitrate is includedin a range of about 1.0 mg/kg to about 50 mg/kg. In some embodiments,ascorbic acid is included in a range of about 1.0 mg/kg to about 25mg/kg. In certain embodiments, L-arginine may also be included in arange of about 2.0 mg/kg to about 50 mg/kg.

In certain embodiments that enhance NO formation in working muscle,sodium nitrite is included in a range of about 30 mg to about 40 mg. Incertain embodiments, sodium nitrate is included in a range of about 250mg to about 300 mg. In certain such embodiments, ascorbic acid isincluded in an amount of about 1000 mg. In certain other embodiments,L-arginine may also be included in an amount of about 1000 mg.

In certain embodiments that function to restore NO homeostasis in theuser, sodium nitrite is included in an amount of about 20 mg. In certainsuch embodiments, sodium nitrate is included in an amount of about 150mg. In certain embodiments, ascorbic acid is included in an amount ofabout 500 mg. In certain other embodiments, L-arginine may also beincluded in an amount of about 500 mg.

The composition can further comprise other materials. In one embodiment,the composition further comprises water. Alternatively or in addition,it can also further comprise other materials. For example, thecomposition may comprise a flavorant, such as a citrus flavor, anon-citrus fruit flavor, an herbal flavor, a vanilla flavor, or achocolate flavor, and other appropriate flavorings.

In other embodiments, the present invention relates to a method ofenhancing cardiovascular performance in a mammal, comprisingadministering to the mammal a composition according to any of theembodiments described herein. In one embodiment, the compositioncomprises a nitrite salt, a nitrate salt, and ascorbic acid.

Any mammal for which enhanced cardiovascular performance is desired canbe the subject of the method. In one embodiment, the mammal is Homosapiens. Other mammals for which enhanced cardiovascular performance maybe desired include, but are not limited to, draft animals, beasts ofburden, animals useful in transportation (e.g., horses), racing animals(e.g., horses or greyhounds), meat animals, wool- or fur-bearinganimals, milk animals, working dogs, and household pets, among others.Enhanced cardiovascular performance can be desired for a person oranimal engaged in physical exertion. In other embodiments, a compositionas described herein may be used as a treatment or prophylaxis for amedical condition characterized by or associated with reduced blood flowto an organ of the body.

Administering the composition can be by any route, such as oral,intravenous, or intraarterial, among others. In one embodiment,administering is by the oral route. In this embodiment, it is desirablethat the components of the composition be dissolved in a neutral- orpleasant-tasting liquid, such as water, flavored water, milk, or fruitjuice, among others. Additionally, the components of the composition maybe in tablet or capsule form and in this form the composition may bedissolvable in liquid. In other embodiments, the composition is providedas a tablet that dissolves when placed in the mouth of a user. In someembodiments, a composition according to any of the embodiments describedherein can be provided in powder, tablet, capsule, gel, aerosol orliquid form.

Any dosage of the components of the composition can be used, providedsuch dosage is safe for the mammal. In one embodiment, administering isof a dosage from about 0.01 mg/kg/day to about 15 mg/kg/day sodiumnitrite, from about 1 mg/kg/day to about 50 mg/kg/day sodium nitrate,and from about 1 mg/kg/day to about 25 mg/kg/day ascorbic acid. If thecomposition comprises L-arginine, administering is of a dosage fromabout 2 mg/kg/day to about 50 mg/kg/day L-arginine.

In one embodiment, the present invention is directed to a composition,comprising from about 40 weight parts to about 1000 weight parts of abotanical nitrate source; from about 20 weight parts to about 500 weightparts of a botanical source of nitrite reduction activity; and fromabout 4 weight parts to about 100 weight parts of a nitrite salt.

A botanical nitrate source is any plant matter, extract of plant matter,or product of plant matter containing nitrate. Generally, it isdesirable that the botanical nitrate source be generally regarded assafe for human or animal consumption. In one embodiment, the botanicalnitrate source is selected from the group consisting of beet root,artichoke, holy basil, gymnema sylvestre, L9H, ashwagandha root, salvia,St. John wort, broccoli, stevia, spinach, gingko, kelp, tribulus,eleuthero, epimedium, eucommia, hawthorn berry, rhodiola, green tea,codonopsys, panax ginseng, astragalus, pine bark, dodder seed,Schisandra, cordyceps, and mixtures thereof. In one particularembodiment, the botanical nitrate source is selected from the groupconsisting of beet root, artichoke, holy basil, gingko, and mixturesthereof.

A botanical source of nitrite reduction activity is any plant matter,extract of plant matter, or product of plant matter, containing nitritereductase enzyme, a compound capable of reducing nitrite, or both.Generally, it is desirable that the botanical source of nitritereduction activity be generally regarded as safe for human or animalconsumption.

In one embodiment, the botanical source of nitrite reduction activity isselected from the group consisting of hawthorn berry, Schisandra, greentea, beet root, pine bark, holy basil, gymnema sylvestre, L9H,ashwagandha root, salvia, St. John wort, broccoli, stevia, spinach,gingko, kelp, tribulus, eleuthero, epimedium, eucommia, rhodiola, greentea, codonopsys, panax ginseng, astragalus, dodder seed, cordyceps,berries, tea, beer, grapes, wine, olive oil, chocolate, cocoa, coffee,walnuts, peanuts, borojo, pomegranates, popcorn, yerba mate, andmixtures thereof. Berries, tea, beer, grapes, wine, olive oil,chocolate, cocoa, coffee, walnuts, peanuts, borojo, pomegranates,popcorn, and yerba mate are known to contain polyphenols, which areknown to have antioxidant properties.

In a particular embodiment, the botanical source of nitrite reductionactivity is selected from the group consisting of hawthorn berry,Schisandra, green tea, beet root, pine bark, and mixtures thereof.

“Hawthorn berry” herein refers to any portion of a plant of the genusCrataegus (for example, Crataegus oxyacantha), such as the berry, leaf,or flower, among others, as well as extracts of any portion thereof. Ina particular embodiment, it refers to the berry of a plant of the genusCrataegus (for example, Crataegus oxyacantha).

“Schisandra” refers to any portion of a plant of the genus Schisandra(for example, S. chinensis and S. rubiflora, among others), such as thefruit, leaf, or flower, among others, as well as extracts of any portionthereof.

A nitrite salt comprising any counterion may be used. Generally, it isdesirable that the nitrite salt be generally regarded as safe for humanor animal consumption. In one embodiment, the nitrite salt is selectedfrom the group consisting of sodium nitrite, potassium nitrite,magnesium nitrite, calcium nitrite, and mixtures thereof. In aparticular embodiment, the nitrite salt is selected from the groupconsisting of sodium nitrite, potassium nitrite, and mixtures thereof.

In addition to the materials described above, the composition canfurther comprise one or more additional materials.

In one embodiment, the composition can further comprise from about 20weight parts to about 500 weight parts L-citrulline.

In one embodiment, the composition can further comprise from about 20weight parts to about 1000 weight parts L-arginine.

The production of NO from L-arginine is a critical cellular functionperformed by nitric oxide synthase (NOS) in most, if not all, organsystems throughout the body. For years, physicians and scientistsassumed simply feeding more substrate L-arginine would be sufficient toenhance NO production. It is becoming increasingly clear that this maynot be the most effective strategy, especially in patients that areinsufficient in NO due to endothelial dysfunction. The effect ofL-arginine on endothelial function in humans is inconsistent. Bydefinition endothelial dysfunction is the inability to produce NO fromL-arginine. This pathway is dysfunctional and inoperative. Providing theenzyme NOS with substrate because of lowered availability of L-argininedoes not appear to be rate limiting since the intracellular levels ofthe amino acid are in the millimolar range (Gold, M. E., P. A. Bush, andL. J. Ignarro, Depletion of arterial L-arginine causes reversibletolerance to endothelium-dependent relaxation. Biochem Biophys ResCommun, 1989. 164(2): p. 714-21), and the enzyme's Michaelis constanst(KM) for substrate is in the micromolar range (2.9 μmol/L) (Bredt, D. S.and S. H. Snyder, Isolation of nitric oxide synthetase, acalmodulin-requiring enzyme. Proc Natl Acad Sci U S A, 1990. 87(2): p.682-5). In contrast, circulating L-arginine measured in plasma ofhealthy humans as well as in plasma of patients with vascular disordersis in the range of 45-100 μmol/L (Boger, R. H. and S. M. Bode-Boger, Theclinical pharmacology of L-arginine. Annu Rev Pharmacol Toxicol, 2001.41: p. 79-99). This is up to 15 to 30-fold higher than the concentrationrequired to saturate the NOS enzyme. This biochemical discrepancy istermed as the “arginine paradox.”

L-Arginine supplementation may be detrimental in some populations. Aclinical trial designed to enhance NO production in humans that havesuffered a heart attack revealed that L-arginine, when added to standardpostinfarction therapies, does not improve vascular stiffnessmeasurements or ejection fraction and may be associated with higherpostinfarction mortality. The study concluded that L-arginine should notbe recommended following acute myocardial infarction (Schulman, S. P.,et al., L-arginine therapy in acute myocardial infarction: the VascularInteraction With Age in Myocardial Infarction (VINTAGE MI) randomizedclinical trial. Jama, 2006. 295(1): p. 58-64).

The vast majority of endogenous arginine synthesis in adult mammals(60%) occurs in the kidney, where citrulline produced by the intestineis extracted from the blood and converted to arginine by the action ofASS and ASL (Windmueller, H. G. and A. E. Spaeth, Source and fate ofcirculating citrulline. Am J Physiol, 1981. 241(6): p. E473-80).Therefore supplying more L-citrulline to the urea cycle will producemore L-arginine to be specifically directed to the nitric oxide pathway;in other words, supplying more L-citrulline provides precursors to theL-arginine nitric oxide pathway while avoiding the negative side effectsof supplementing L-arginine directly.

In one embodiment, the composition can further comprise from about 0.2weight parts to about 5 weight parts vitamin B12. The vitamin B12 can bein any form of cobalamin. In one embodiment, the vitamin B12 is in aform selected from the group consisting of methylcobalamin,cyanocobalamin, and mixtures thereof.

In one embodiment, the composition can further comprise from about 20weight parts to about 500 weight parts vitamin C. The vitamin C can bein any form of ascorbate or ascorbic acid. In one embodiment, thevitamin C is in a form selected from the group consisting of magnesiumascorbate, sodium ascorbate, potassium ascorbate, ascorbicacid, andmixtures thereof.

In one embodiment, the composition can further comprise from about fromabout 20 weight parts to about 500 weight parts of a nitrate saltselected from the group consisting of sodium nitrate, potassium nitrate,and mixtures thereof.

In addition to the materials described above, the composition canfurther comprise one or more additional materials suitable for formingthe composition into a vehicle deliverable for human or animalconsumption. Such one or more additional materials include, but are notlimited to, binders, flavorants, colorants, sweeteners, adujvants, andexcipients, among others.

In one embodiment, the composition can further comprise from about 50weight parts to about 1500 weight parts of one or more other ingredientsselected from the group consisting of mannitol, xylitol, sorbitol, othersugar alcohols, cellulose, cellulose esters, cellulose ethers, othermodified celluloses, starch, modified starches, other polysaccharides,oligosaccharides, disaccharides, saccharides, gelatin,polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants,colorants, magnesium stearate, other antiadherent agents, other stearatesalts, sweeteners, silica, and other lubricants. These one or moreingredients can act as one or more of binders, flavorants, colorants,sweeteners, antiadherents, or lubricants, among other functions.

In one particular embodiment, the composition can be as follows:

the botanical nitrate source is selected from the group consisting ofbeet root, artichoke, holy basil, gingko, and mixtures thereof and ispresent at about 200 weight parts;

the botanical source of nitrite reduction activity is selected from thegroup consisting of hawthorn berry, Schisandra, green tea, beet root,pine bark, and mixtures thereof and is present at about 100 weightparts; and

the nitrite salt is selected from the group consisting of sodiumnitrite, potassium nitrite, and mixtures thereof and is present at about20 weight parts;

and the composition further comprises:

about 100 weight parts L-citrulline;

about 1 weight part vitamin B12 in a form selected from the groupconsisting of methylcobalamin, cyanocobalamin, and mixtures thereof;

about 100 weight parts vitamin C in a form selected from the groupconsisting of magnesium ascorbate, ascorbic acid, and mixtures thereof;and from about 50 weight parts to about 1500 weight parts of one or moreother ingredients selected from the group consisting of mannitol,xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters,cellulose ethers, other modified celluloses, starch, modified starches,other polysaccharides, oligosaccharides, disaccharides, saccharides,gelatin, polyvinylpyrrolidone, polyethylene glycol, other binders,flavorants, colorants, magnesium stearate, other antiadherent agents,other stearate salts, sweeteners, silica, and other lubricants.

The composition can be formulated, using techniques known in the art,into any vehicle suitable for human consumption. For example, thecomposition can be formulated as a powder dissolvable or suspendable ina potable beverage, a soft food, or both; as an ingredient that can bebaked into a baked cookie, cracker, or bar; a tablet or capsule that canbe swallowed; or a lozenge dissolvable in the mouth; among others. Inone embodiment, the composition can be in a form of a lozengedissolvable in the mouth. The lozenge can have a weight from about 600mg to about 2000 mg.

In one embodiment, the present invention is directed to a method ofreducing a patient's triglyceride level, comprising administering to thepatient a composition in a form of a lozenge dissolvable in the mouth,the composition comprising from about 40 weight parts to about 1000weight parts of a botanical nitrate source; from about 20 weight partsto about 500 weight parts of a botanical source of nitrite reductionactivity; from about 20 weight parts to about 500 weight partsL-citrulline; and from about 4 weight parts to about 100 weight parts ofa nitrite salt.

The composition and its components can be as described above. Also, theformulation of the composition as a lozenge can be as described above.

In one embodiment, 1 weight part is 1 mg.

In a particular embodiment, the administered composition can be asfollows:

the botanical nitrate source is selected from the group consisting ofbeet root, artichoke, holy basil, gingko, and mixtures thereof and ispresent at about 200 mg;

the botanical source of nitrite reduction activity is selected from thegroup consisting of hawthorn berry, Schisandra, green tea, beet root,pine bark, and mixtures thereof and is present at about 100 mg; and

the nitrite salt is selected from the group consisting of sodiumnitrite, potassium nitrite, and mixtures thereof and is present at about20 mg;

and the composition further comprises:

about 100 mg L-citrulline;

about 1000 μg vitamin B12 in a form selected from the group consistingof methylcobalamin, cyanocobalamin, and mixtures thereof;

about 100 mg vitamin C in a form selected from the group consisting ofmagnesium ascorbate, ascorbic acid, and mixtures thereof; and

from about 50 mg to about 1500 mg of one or more other ingredientsselected from the group consisting of mannitol, xylitol, sorbitol, othersugar alcohols, cellulose, cellulose esters, cellulose ethers, othermodified celluloses, starch, modified starches, other polysaccharides,oligosaccharides, disaccharides, saccharides, gelatin,polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants,colorants, magnesium stearate, other antiadherent agents, other stearatesalts, sweeteners, silica, and other lubricants.

The composition can be administered according to any dosing regimen.Particular details of how administering is to be performed are withinthe ability of the person of ordinary skill in the art having thebenefit of the present disclosure. In one embodiment, the administeringis performed once or twice daily.

In one embodiment, the present invention is directed to a method ofreducing a patient's C-reactive protein level, comprising administeringto the patient a composition in a form of a lozenge dissolvable in themouth, the composition comprising from about 40 weight parts to about1000 weight parts of a botanical nitrate source; from about 20 weightparts to about 500 weight parts of a botanical source of nitritereduction activity; from about 20 weight parts to about 500 weight partsL-citrulline; and from about 4 weight parts to about 100 weight parts ofa nitrite salt.

The composition and its components can be as described above. Also, theformulation of the composition as a lozenge can be as described above.

In one embodiment, 1 weight part is 1 mg.

In a particular embodiment, the administered composition can be asfollows:

the botanical nitrate source is selected from the group consisting ofbeet root, artichoke, holy basil, gingko, and mixtures thereof and ispresent at about 200 mg;

the botanical source of nitrite reduction activity is selected from thegroup consisting of hawthorn berry, Schisandra, green tea, beet root,pine bark, and mixtures thereof and is present at about 100 mg; and

the nitrite salt is selected from the group consisting of sodiumnitrite, potassium nitrite, and mixtures thereof and is present at about20 mg;

and the composition further comprises:

about 100 mg L-citrulline;

about 1000 μg vitamin B12 in a form selected from the group consistingof methylcobalamin, cyanocobalamin, and mixtures thereof;

about 100 mg vitamin C in a form selected from the group consisting ofmagnesium ascorbate, ascorbic acid, and mixtures thereof; and

from about 50 mg to about 1500 mg of one or more other ingredientsselected from the group consisting of mannitol, xylitol, sorbitol, othersugar alcohols, cellulose, cellulose esters, cellulose ethers, othermodified celluloses, starch, modified starches, other polysaccharides,oligosaccharides, disaccharides, saccharides, gelatin,polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants,colorants, magnesium stearate, other antiadherent agents, other stearatesalts, sweeteners, silica, and other lubricants.

The composition can be administered according to any dosing regimen.Particular details of how administering is to be performed are withinthe ability of the person of ordinary skill in the art having thebenefit of the present disclosure. In one embodiment, the administeringis performed once or twice daily.

The following examples are included to demonstrate certain embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES Example 1. Use of Sodium Nitrite as an Alternate Source ofNitric Oxide in Muscle Training

Exercising muscle demands increased blood flow in order to maintainsufficient nutrients and oxygen for metabolism. Nitric oxide is thebody's most potent vasodilator. Nitric oxide is produced in the body bythe enzyme nitric oxide synthase (NOS). NOS enzymes produce .NO bycatalyzing a five electron oxidation of a guanidino nitrogen ofL-arginine (L-Arg). Oxidation of L-Arg to L-citrulline occurs via twosuccessive monooxygenation reactions producing

hydroxy L-arginine as an intermediate. Two moles of O2 and 1.5 moles ofNADPH are consumed per mole of NO formed (Liu, Q. and G. S. S., Bindingsites of nitric oxide synthases. Methods Enzymol, 1996. 268: p.311-324). NOS enzymes are the only enzymes known to simultaneouslyrequire five bound cofactors/prosthetic groups: FAD, FMN, heme,tetrahydrobiopterin (BH4) and Ca²⁺— calmodulin (CaM). All NOS isozymesare catalytically self-sufficient provided all required substrates andco-factors are available. CaM binding to nNOS has been shown to regulatecatalytic activity by triggering electron flux from FMN to heme, therebycoupling the oxygenase and reductase domains. CaM also facilitates NADPHdependent reduction of cytochrome c and ferricyanide in BH₄ and hemedepleted nNOS. If any of the co-factors become limiting, then NOproduction from NOS shuts down, and in many cases NOS then producessuperoxide instead. This is indeed a very complex and coordinated effortto enzymatically produce NO which normally proceeds very efficiently.However, in disease characterized by oxidative stress where cofactorsbecome oxidized, NOS uncoupling, or conditions of hypoxia where oxygenis limiting, this process can no longer maintain NO production.Therefore there has to be an alternate route to NO production. It ishighly unlikely that Nature devised such a sophisticated mechanism of NOproduction as a sole source of a critical molecule. Nitrite reductionthen acts as a backup system to the NOS system. Part of this may occurthrough nitrite reduction during low oxygen availability. Nitritesupplementation can then support NO production during exercise whenenzymatic NO production is shut down.

Nitrite reduction to NO can occur via a simple mechanism. The 1-electronreduction of nitrite can occur by ferrous heme proteins (or any redoxactive metal) and an electron donor through the following reaction:

NO₂ ⁻+Fe^((II))+H⁺↔NO+Fe^((III))+OH⁻

This is the same biologically active NO as that produced by NOS justinstead of using L-arg as the substrate, nitrite is used. Therefore forthis to occur, the tissues or biological compartment must have asufficient pool of nitrite stored. Nitrite supplementation may thereforeact as a protective measure to compensate for insufficient NOS activityunder conditions of hypoxia such as during anaerobic metabolism duringexercise or muscle training. Nitrite contributes to whole body NOproduction and homeostasis. Considerable published support for thistheory derives from the following facts: NO produced from nitrite in theupper intestine is up to 10,000 times the concentrations that occur intissues from enzymatic synthesis (McKnight, G. M., et al., Chemicalsynthesis of nitric oxide in the stomach from dietary nitrate in humans.Gut, 1997. 40(2): p. 211-214), nitrite can act as a circulating NO donor(Dejam, A., et al., Emerging role of nitrite in human biology. BloodCells Mol Dis, 2004. 32(3): p. 423-429) and nitrite can itself performmany actions previously attributable to NO (Gladwin, M. T., et al., Theemerging biology of the nitrite anion. Nature Chemical Biology, 2005.1(6): p. 308-314) without the intermediacy of NO (Bryan, N. S., et al.,Nitrite is a signaling molecule and regulator of gene expression inmammalian tissues. Nature Chemical Biology, 2005. 1(5): p. 290-297).While L-arginine supplementation may provide moderate amounts of NOprior to workout, during a workout, this system becomes inefficient andvery little NO from L-arginine can be produced due to lack of oxygensubstrate. Therefore NO from nitrite provides an alternate mechanism tomaintain NO production during exercise. Supplemental nitrite taken 15-20minutes prior to workout can titrate up tissue and muscle nitriteconcentrations in order to produce NO locally during exercise andtherefore enhance blood flow and performance

Nitrite Reduction to NO is an Oxygen Sensitive Process

Much of the recent focus on nitrite physiology is due to its ability tobe reduced to NO during ischemic or hypoxic events (Lundberg, J. O. andE. Weitzberg, NO generation from nitrite and its role in vascularcontrol. Arterioscler Thromb Vasc Biol, 2005. 25(5): p. 915-22; Bryan,N. S., Nitrite in nitric oxide biology: Cause or consequence? Asystems-based review. Free Radic Biol Med, 2006. 41(5): p. 691-701;Bryan, N. S., et al., Cellular Targets and Mechanisms ofNitros(yl)ation: An Insight into Their Nature and Kinetics in vivo.Proc. Natl. Acad Sci. USA, 2004. 101(12): p. 4308-4313). Nitritereduction to NO under aerobic and anaerobic conditions usingchemiluminescent detection of free NO has been quantified andcharacterized. Under aerobic conditions, established by continuoussample purging with air, NO production by blood-free tissues and RBCsfrom nitrite was minimal and fleeting. However, switching the purge gasto N₂ (i.e., hypoxia) acutely enhanced tissue NO formation from NO₂ ⁻(FIG. 1, left). Hypoxic tissue NO₂ ⁻ reduction enhanced tissue NOformation from NO²⁻ (FIG. 1, left). Hypoxic tissue NO²⁻ reductionexhibited compartment-specific properties (initial kinetics, amount,duration) and was most dramatic and sustained in liver homogenate. Inaddition to liver, all tissues sampled were capable of detecting animposed decrease in O₂ tension and transducing this information into apotentiation of tissue NO formation from NO²⁻, as demonstrated by themarked, tissue-selective increases in NO production from NO²⁻ observedunder aerobic (21% O₂) vs. hypoxic (N₂) conditions (FIG. 1, right).Heart, liver, skeletal (gastrocnemius) muscle, and aorta exhibited thegreatest capacity for NO₂ ⁻ reduction to NO during O₂ deprivation (FIG.1, right inset). These data demonstrate an intrinsic ability of tissuesto sense the graded diminution of ambient O₂ and transduce thisinformation into the subsequent production of NO from NO₂ ⁻. Thisfinding suggests that various tissues might auto-regulate their bloodflow in disease states with a pathological component of O₂ insufficiencythrough their capacity for hypoxic NO₂ ⁻ reduction to NO providedsufficient nitrite exist.

Example 2. Nitrite Can Protect Tissues from Ischemia-Reperfusion Injury

There is a growing appreciation that nitrite therapy may provide benefitfrom I/R injury (Dezfulian, Raat et al. 2007). However, there are nodata on the effects of nitrite insufficiency in the setting of I/Rinjury. Analysis of several different standard rodent chows revealedthat Purina 5001 contains the highest concentrations of NOx (104.3±4.7pmol/g nitrite and 6275±50.7 pmol/g nitrate) and nitroso as compared toany other standard rodent chow analyzed. Therefore this diet was used incomparison to mice fed a purified amino acid diet, the diet lowest inNOx (20.5±0.7 pmol/g nitrite and 503.1±17.9 pmol/g nitrate) but with thesame L-arginine content. In order to reveal the biochemical andphysiological effects of dietary nitrite insufficiency, mice were fed astandard rodent chow (Purina 5001) for 9 weeks and then switched to apurified amino acid diet low in nitrite and nitrate (Harlan TD99366) for7 days. Control mice were fed Purina 5001 for 10 weeks. Consistent withan earlier report (Bryan, Fernandez et al. 2005), the low NOx dietsignificantly decreased plasma and heart steady-state nitrite andnitrate concentrations which could be restored by the addition of 50mg/L nitrite in the drinking water for 1 week (FIG. 2A-B). Blood andtissue nitroso products have been shown to preserve NO bioactivity(Stamler, Simon et al. 1992) and protein nitrosation modification confercGMP-independent NO signalling events (Stamler, Lamas et al. 2001).Changes in dietary nitrite consumption affect cellular signaling events(Bryan, Fernandez et al. 2005). Mice fed a low NOx diet for 1 weekdemonstrated a significant reduction in plasma and heart nitroso levelscompared to mice fed standard chow, which could be replenished andincreased with 50 mg/L nitrite in the drinking water for 1 week (FIG.2C). Nitrosyl-heme products (FIG. 2D) were also reduced in the mice feda low NOx diet and replenished by nitrite supplementation in thedrinking water. These data reveal that changes in dietary nitrite and/ornitrate consumption can affect steady state concentrations of blood andtissue NO products/metabolites commonly used to assess NO production.

Whether dietary restriction of nitrite affected the severity of cardiacischemia-reperfusion (I/R) injury was determined. The decrease in steadystate nitrite concentrations in blood and heart was found tosignificantly exacerbate myocardial injury (FIG. 2E). The mice fed a lowNOx diet displayed a 59% increase in infarct relative to the area atrisk (AAR) compared to mice fed a standard chow. To ensure the observedeffect was dependent upon NOx intake, and not due to an alteration inthe nutritional value of the low NOx diet, a subset of mice on the lowNOx diet were given 50 mg/L sodium nitrite ad libitum in the drinkingwater to restore steady state concentrations of blood and tissuenitrite. Nitrite supplementation in animals on the low NOx diet reversedthe increased myocardial infarct size by 57%. Additionally, mice fed thelow NOx diet displayed a higher mortality rate (57.7% survival) 24 hourspost-myocardial infarction than mice on the standard rodent chow (70.6%survival). Likewise, survival improved in mice on the low NOx diet withnitrite-supplemented drinking water to 76.9%. Since nitrite is derivedboth from diet and oxidation of enzymatic NO production from NOS,potential compensatory changes in NOS expression following one week lowNOx intake were investigated. Western blot analysis of myocardial tissuelysate revealed no significant alterations in NOS expression (eNOS,nNOS, and iNOS) (FIG. 2F). These data clearly suggest that the increasedinjury is due specifically to changes in steady state concentrations ofplasma and heart nitrite as a result of decreased dietary NOxconsumption and not from changes in enzymatic NO production.

Duranski et al. recently demonstrated that bolus addition of nitriteprior to reperfusion significantly protects the heart and liver fromischemia/reperfusion damage in an in vivo model (Duranski, M. R., etal., Cytoprotective effects of nitrite during in vivoischemia-reperfusion of the heart and liver. J Clin Invest, 2005.115(5): p. 1232-1240). During the ischemic event, NOS is inactive sinceoxygen, a necessary co-factor, has been depleted. It is believed thatnitrite is reduced to NO to compensate for the insufficient NOS derivedNO. These data are very important because they helped us to recognizethat the application of exogenous nitrite has profound effects.Endurance training is known to cause ischemic organ damage in suchorgans as the gut and kidneys due to the diversion of blood flow fromthese organs to supply working muscles. This is a significant problem inmarathon runners. Nitrite may provide a valuable nutrient to theseathletes as a pre-workout or pre-marathon supplement to protect fromischemic injury during the event. Nitrite may then serve multiplepurposes in the setting of myocardial ischemia/reperfusion. First, bytitrating up tissue concentrations of nitrite when it is administeredjust prior to reperfusion, one can protect the heart fromischemia/reperfusion. Second, acute nitrite administration may initiatea signaling cascade that results in the upregulation of other protectiveproteins which afford protection hours later.

Nitrite has been shown to be protective in both the heart and the liverfollowing ischemia/reperfusion (Webb, A., et al., Reduction of nitriteto nitric oxide during ischemia protects against myocardialischemia-reperfusion damage. Proc Natl Acad Sci USA, 2004.101(13683-13688); Duranski, M. R., et al., Cytoprotective effects ofnitrite during in vivo ischemia-reperfusion of the heart and liver. JClin Invest, 2005. 115(5): p. 1232-1240). It is speculated that nitriteis reduced to NO under ischemic conditions to provide an alternatesource of NO when NOS is inactive due to decreased substrate deliveryand decreased oxygen saturation. To better understand the fate ofnitrite during ischemia and reperfusion, a time course of nitritemetabolism both after ischemia and during reperfusion was conducted. Asshown in FIG. 3, nitrite is consumed in the heart tissue during 30minutes of ischemia but is unaffected in the plasma. The consumption ofnitrite appears to lead to a concomitant increase in cardiac nitrosoproducts (FIG. 3B). Nitrite can form nitrosothiols in a first orderreaction requiring heme and thiols and can also be reduced to NO underanaerobic conditions (Bryan, N. S., et al., Nitrite is a signalingmolecule and regulator of gene expression in mammalian tissues. Nat ChemBiol, 2005. 1(5): p. 290-7). During reperfusion, nitrite is graduallyincreased and restored whereby tissue nitroso decompose during thereperfusion phase (FIG. 3). Without being bound by a particular theory,the inventor proposes that nitrite serves two functions in the settingof ischemia reperfusion. It may first serve as a NOS-independent sourceof NO by which nitrite is reduced to NO under ischemic conditions whenNOS is inactive. Secondly, nitrite may react with critical thiols toform nitrosothiols. It is possible that this nitroso modification actsas a reversible protective shield which reduces irreversible oxidationduring the oxidative burst of reperfusion. Aside from “capping” criticalthiols from oxidation, and without being bound by a particular theory,the inventor proposes that the nitroso products can then release the NO+moiety during the reperfusion phase and act an a redox sensitive NOdonor (Hogg, N., Biological chemistry and clinical potential ofS-nitrosothiols. Free Radic Biol Med, 2000. 28(10): p. 1478-86).Biochemical data support this notion by the increase in nitroso at theexpense of nitrite followed by the decay of nitroso over time duringreperfusion. Therefore adding supplemental nitrite can increase plasmaand tissue nitrite but also lead to an increase in steady state levelsof nitroso and thereby afford protection during ischemia/reperfusion. Onthe contrary, nitrite insufficiency leads to increased injury becausethere is not enough stored in blood or tissue to perform these actions.

Enzymatic NO insufficiency is a hallmark of a number of diseasesincluding cardiovascular disease. To test the hypothesis that dietarynitrite can compensate for NOS dysfunction, the above experiments wererepeated in eNOS−/− mice. The mice were fed standard rodent chow or lowNOx diet. As shown in FIG. 4, eNOS−/− mice revealed lower plasma nitriteconcentrations consistent with earlier findings (Kleinbongard, P., etal., Plasma nitrite reflects constitutive nitric oxide synthase activityin mammals. Free Radical Biology & Medicine, 2003. 35(7): p. 790-796)but there is no significant difference in cardiac nitrite revealing thatblood markers do not accurately reflect tissue status (Bryan, N. S.,Nitrite in nitric oxide biology: Cause or consequence? A systems-basedreview. Free Radic Biol Med, 2006. 41(5): p. 691-701). Plasma nitritecould be further decreased by feeding eNOS−/− mice a low NOx dietdemonstrating that plasma nitrite is a reflection of both NOS and diet.Feeding low NOx diet to eNOS−/− mice completely eliminated steady stateconcentrations of plasma nitroso without any significant effect oncardiac nitroso. Supplementation of 50 mg/L nitrite in the drinkingwater for 7 days restores plasma nitrite in eNOS−/− to control levelsand increases both plasma and cardiac nitroso to above C57 controllevels.

Mice deficient in eNOS have increased injury to ischemia/reperfusioninsult (Jones, S. P., et al., Myocardial ischemia-reperfusion injury isexacerbated in absence of endothelial cell nitric oxide synthase. Am JPhysiol, 1999. 276(5 Pt 2): p. H1567-73) and data shown above revealthese mice also have reduced nitrite and nitroso compared to C57 wildtype. To investigate if dietary nitrite can benefit eNOS−/− mice,eNOS−/− mice on low NOx diet±50 mg/L nitrite in drinking water weresubjected to 30 minutes ischemia and 24 hour reperfusion as above. Thesemice are from myocardial ischemia/reperfusion injury suggesting thatdietary nitrite supplementation can provide benefit under conditions ofdysfunctional NOS.

Example 3. Nitrite Can be Used to Delay or Prevent the Onset andDevelopment of Atherosclerosis

Initial studies in the characterization of the LDb mouse reveal thatthey have diminished blood and tissue nitrite and nitroso levels with nodifference in nitrate at 11 months when atherosclerosis is welldeveloped. These data indicate that there is deficiency in bioavailableNO and nitrite. Steady state tissue nitroso levels are also decreasedsuggesting a dysregulation of protein nitrosation and therefore providethe rationale and justification for early intervention of dietarynitrite supplementation on restoring NO-nitroso redox and on theprogression of atherosclerosis.

In order to demonstrate the utility of supplemental nitrite in affectingthe progression of atherosclerosis, a high fat diet was fed to 8 femaleLDb mice for 12 weeks. Four of the mice received nitrite free water andthe other 4 mice were supplemented with 50 mg/L nitrite throughout the12 weeks on high fat diet. Although the LDb mice spontaneously developatherosclerosis on normal rodent chow, the addition of a high fat dietwill accelerate the process from 8 months to 12 weeks. At the time ofsacrifice, plasma was collected for nitrite, nitrate determination aswell as lipid profile determination. As shown in FIGS. 5A-B, theresignificantly more circulating nitrite and nitrate in the nitrite fedmice than the nitrite free water group. The nitrite fed group had 20%less lesion formation on the abdominal aorta than the control group fedhigh fat diet with nitrite free water (FIG. 5C). These data demonstratethat nitrite supplementation can inhibit the progression ofatherosclerosis in the female LDb mice using a high fat diet.

Example 4. Specific Formulation for Extending Biological Half Life OfNitrite

Aim: To develop specific formulation that will extend the circulatinghalf life of nitrite from 110 seconds to 45-60 minutes.

Methods: Since nitrite is derived from NO oxidation, diet, and from thereduction of nitrate in the human body, to enhance nitritebioavailability, substrates from all 3 pathways were included:L-arginine to enhance NO production from nitric oxide synthase whichwill subsequently produce nitrite; sodium nitrite to increase acutecirculating nitrite concentrations; ascorbic acid to inhibit endogenousnitrosation reactions in the stomach; and sodium nitrate to provide aslow release source of nitrite.

This specific formulation was compared to 3 g L-arginine that ismarketed commercially to enhance NO production. An intravenous line wasobtained by the inventor on himself with 21 gauge infusion set needleand blood collected. The first 5 ml of blood was discarded. Blood wasthen collected at baseline. Then the prescribed formulation wasdissolved in 50 ml of water and taken orally. A timer was started andblood was sampled for analysis at 1 minute and every 2 minutes for 60minutes.

Results: Direct analysis of the volunteer's blood revealed that the oralformulation increases blood nitrite within 3 minutes and reaches amaximum 9 minutes (FIG. 6). This represents a 20 fold increase in plasmanitrite that lasts for 50 minutes. Plasma nitrate continues to risethroughout the course of the experiment. L-arginine in combination withNAD and ascorbic acid did not significantly affect plasma nitrite ornitrate concentrations (FIG. 7).

Conclusions: The specific formulation developed can increase plasmanitrite to therapeutic levels within 3 minutes of ingestion and canmaintain therapeutic levels until 50 minutes after drinking. Thisrepresents a novel formulation whereby this product can be givenimmediately upon patient presentation of ischemic episode that willmaintain the protective nitrite levels for up to 1 hour. There is agolden hour in clinical medicine whereby the patient survival andoutcome from ischemic episodes greatly declines. The safety of nitriteand nitrate at these doses is well established and therefore theformulations according to several embodiments discussed above representa safe, novel use for oral nitrite as a cardioprotective agent.

Example 5. Specific Formulations

Disclosed herein is an immediate and extended release form of nitriteextending the biological half life from hundreds of seconds to minutesand hours. Among others, two possible applications for this technologymay be as a revolutionary nitric oxide based supplement for the workoutindustry and a daily supplement to restore NO homeostasis in the agingpopulation. Below is a range of effective doses of each ingredient.

Workout Supplement and Daily Supplement

Sodium nitrite (0.01 mg/kg-15 mg/kg: fatal dose is 22-23 mg/kg inhumans) Sodium nitrate (1.0 mg/kg-50 mg/kg; Poisoning in man may resultfrom a total oral daily dose in excess of 4 g or from a single dose ofmore than 1 g. 8 g may be fatal and 13-15 g are generally fatal(Sollmann, 1957). Although natural sources of nitrate are available, theconcentrations are sufficiently low that the volume (or mass) that wouldneed to be consumed to provide the same degree of supplementation ascompared to several embodiments disclosed herein would be prohibitivelylarge. For example, one liter of beetroot juice contains about 2.79 g ofnitrate.

Ascorbic acid (1 mg/kg-25 mg/kg)

L-arginine (2 mg/kg-50 mg/kg)

In one embodiment, a workout supplement formulation to enhance NOformation in working muscle consists, consists essentially of orcomprises:

40 mg sodium nitrite

250 mg sodium nitrate

1000 mg ascorbic acid

1000 mg L-arginine

In another embodiment, a workout supplement formulation to enhance NOformation in working muscle consists, consists essentially of orcomprises:

30 mg sodium nitrite

300 mg sodium nitrate

1000 mg ascorbic acid

1000 mg L-arginine

In one embodiment, a daily supplement formulation to restore NOhomeostasis consists, consists essentially of or comprises:

20 mg sodium nitrite

150 mg sodium nitrate

500 mg ascorbic acid

500 mg L-arginine

FIG. 6 shows blood nitrite and nitrate levels from 0 min to 60 min afteringestion of an oral formulation containing 30 mg sodium nitrite, 300 mgsodium nitrate, 1000 mg ascorbic acid, and 1000 mg L-arginine in a humanvolunteer. In one embodiment, the formulation consists, consistsessentially of or comprises 30 mg sodium nitrite, 300 mg sodium nitrate,1000 mg ascorbic acid, and 1000 mg L-arginine.

Example 6. NO Generation without NO Synthase

In 1994 two groups independently presented evidence for generation of NOin the stomach resulting from the acidic reduction of inorganic nitrite(Benjamin, N., F. O'Driscoll, et al. (1994). “Stomach NO synthesis.”Nature 368(6471): 502; Lundberg, J. O., E. Weitzberg, et al. (1994).“Intragastric nitric oxide production in humans: measurements inexpelled air.” Gut 35(11): 1543-6). Benjamin and colleagues demonstratedthat the antibacterial effects of acid alone was markedly enhanced byaddition of nitrite which is present in saliva whereas Lundberg andcolleagues could measure high levels of NO in expelled air from thestomach in humans. These levels were abolished after pretreatment with aproton pump inhibitor and markedly increased after ingestion of nitrate,showing the importance of both luminal pH and the conversion of nitrateto nitrite for stomach NO generation. These were the first reports of NOsynthase-independent formation of NO in vivo.

In the classical NO synthase pathway, NO is formed by oxidation of theguanidino nitrogen of L-arginine with molecular oxygen as the electronacceptor (Moncada, S. and A. Higgs (1993). “The L-arginine-nitric oxidepathway.” N Engl J Med 329(27): 2002-12). This complex reaction iscatalysed by specific heme-containing enzymes, the NO synthases, and thereaction requires several co-factors. The alternative pathway wasfundamentally different; instead of L-arginine it used the simpleinorganic anions nitrate (NO3—) and nitrite (NO2—) as substrates in astepwise reduction process that did not require NO synthase or multipleco-factors. The biochemical pathway and biological effects of nitratereduction to nitrite and further on to NO in the gastrointestinal tracthave now been further characterized (Lundberg, J. O., E. Weitzberg, etal. (2004). “Nitrate, bacteria and human health.” Nat Rev Microbiol2(7): 593-602). Oral commensal bacteria are essential for the first stepin the nitrate-nitrite-NO pathway since they are responsible for thereduction of the higher nitrogen oxide nitrate to form nitrite. It wasknown from the literature that the salivary glands extract nitrate fromplasma but the reason for this active process was not explained. Thisactive process leads to levels of salivary nitrate that are 10-20 foldhigher than in plasma. Oral facultative anaerobic bacteria residingmainly in the crypts of the tongue, then reduce nitrate to nitrite bythe action of nitrate reductase enzymes (Spiegelhalder, B., G.Eisenbrand, et al. (1976). “Influence of dietary nitrate on nitritecontent of human saliva: possible relevance to in vivo formation ofN-nitroso compounds.” Food Cosmet Toxicol 14: 545-548; Duncan, C., H.Dougall, et al. (1995). “Chemical generation of nitric oxide in themouth from the enterosalivary circulation of dietary nitrate [seecomments].” Nat Med 1(6): 546-51). These bacteria use nitrate as analternative electron acceptor to gain ATP in the absence of oxygen. Thishighly effective bacterial nitrate reduction results in salivary levelsof nitrite that are 1000-fold higher than those found in plasma(Lundberg, J. O. and M. Govoni (2004). “Inorganic nitrate is a possiblesource for systemic generation of nitric oxide.” Free Radic Biol Med37(3): 395-400). When nitrite-rich saliva meets the acidic gastricjuice, nitrite is protonated to form nitrous acid (HNO2) which thendecomposes to NO and a variety of other nitrogen oxides (Benjamin,O'Driscoll et al. 1994; Lundberg, Weitzberg et al. 1994). It is nowestablished that oral commensal bacteria are pivotal in gastric NOformation, and gastric NO levels are consistently low in animals rearedunder complete germ-free conditions (Sobko, T., C. Reinders, et al.(2004). “Gastrointestinal nitric oxide generation in germ-free andconventional rats.” Am J Physiol Gastrointest Liver Physiol 287(5):G993-7). If the oral flora is selectively removed by topical treatmentwith an antibacterial mouthwash, the gastric NO levels decreasedrastically (Petersson, J. (2008). Nitrate, nitrite and nitric oxide ingastric mucosal defense. Uppsala University. Uppsala, Sweden). Thispathway is now known to contribute to at least half of the endogenous NOmetabolism in humans.

A Novel Nitric Oxide System: This recognition now provides a newparadigm for restoring NO homeostasis that is vastly different than theNOS system. The stepwise reduction of nitrate to nitrite to nitric oxideis, by necessity, an inefficient process by which each step yields a3-log lower concentration of product than substrate (Jansson, E. A., L.Huang, et al. (2008). “A mammalian functional nitrate reductase thatregulates nitrite and nitric oxide homeostasis.” Nat Chem Biol 4(7):411-7). Steady state levels of nitrate and nitrite in healthy humans is20-40 μM and ˜0.5 μM respectively. The relative ratio of nitrate tonitrite in blood corroborates this 3-log reduction efficacy of nitrateto nitrite. Given this same efficiency for nitrite, one might expect ˜5nM NO production from reduction of physiological concentrations ofnitrite, which would certainly elicit a physiological response. However,we have found that oxygen is a potent inhibitor of nitrite reductionwith 80% inhibition at 0.5% oxygen (Feelisch, M., B. O. Fernandez, etal. (2008). “Tissue Processing of Nitrite in Hypoxia: An IntricateInterplay of Nitric Oxide-Generating and -Scavenging Systems.” J BiolChem 283(49): 33927-34). In vitro measurements indicate only 0.01% ofnitrite is converted to NO in completely anaerobic and anoxic tissuehomogenates and actually much less at physiological concentrations ofoxygen (Bryan, N. S., B. O. Fernandez, et al. (2005). “Nitrite is asignaling molecule and regulator of gene expression in mammaliantissues.” Nat Chem Biol 1(5): 290-7). Since humans with knowncardiovascular risk factors and endothelial dysfunction have reducedplasma and presumably tissue levels of nitrite (Kleinbongard, P., A.Dejam, et al. (2006). “Plasma nitrite concentrations reflect the degreeof endothelial dysfunction in humans.” Free Radic Biol Med 40(2):295-302), this reservoir of NO activity is further compromised andtherefore unable to be utilized for NO production by inherent biologicalsystems. As a result, in order to get an appreciable amount of NO fromthis pathway, pharmacological doses of nitrate and nitrite are neededwhen relying simply on inherent biological systems for the reduction.

Although both nitrite and nitrate are naturally occurring molecules andare part of our food supply (Hord, N. G., Y. Tang, et al. (2009). “Foodsources of nitrates and nitrites: the physiologic context for potentialhealth benefits.” Am J Clin Nutr 90(1): 1-10), they are not withouttoxicity. We have created a system whereby we can enhance thenitrate-nitrite-NO reduction efficacy at each step which justifies theuse of physiological concentrations of nitrate and nitrite as aprovision of NO which can overcome the body's inability to generate NOfrom L-arginine.

Remarkably, we have identified natural products that are part of ournormal diet which can enhance this pathway. We recently completed anationwide survey of over 400 food products including meats andvegetables, both organically and conventionally grown and screened over100 herbs that have been used for many years in traditional medicalpractices in Europe and Asia. The results of our study have revealedproducts that are enriched in nitrate (e.g., beet root) that provide thesubstrate nitrite in the pathway. We have also uncovered a select fewherbs with very robust nitrite reductase activity that is unaffected byoxygen (e.g., Hawthorne berry).

As illustrated in FIG. 8. the endogenous production of nitric oxide byNOS leads to the rapid oxidation to nitrite by molecular oxygen andplasma proteins such as ceruloplasmin. Alternatively, NO reacts directlywith oxyheme-proteins such as hemoglobin and is oxidized to nitrate. Wenow have a system to reverse this process and regenerate NO from theseoxidative end-products, nitrite and nitrate. The introduction of beetand Hawthorne provide a very robust system for generating NO that isnovel and innovative and allows us to use much lower physiologicalconcentrations of nitrite rather than pharmacological therapeutic doses.

We recently published our initial results from these investigationsusing Traditional Chinese Medicines (TCM) (Tang, Y., H. Garg, et al.(2009). “Nitric oxide bioactivity of traditional Chinese medicines usedfor cardiovascular indications.” Free Radic Biol Med 47(6): 835-40).Since many of these TCMs are used primarily for cardiovascularindications characterized by NO insufficiency, we hypothesized thatsome, if not all, of these TCMs have a robust NO bioactivity that mayact to restore NO homeostasis. The results from our study reveal thatthe herbs tested contained varying levels of nitrite and nitrate andvarying nitrite reductase activity. Several of the herbal extractscontain a nitrite reductase activity greater by 1000 times than that ofbiological tissues (Feelisch, Fernandez et al. 2008). We have discoveredthat Hawthorn berry contains an active nitrite reductase that is 10times more active than the Chinese herbs we tested.

We concluded that repletion of biological nitrite and nitrate by theseextracts and providing a natural system for NO generation in bothendothelium-dependent and -independent mechanisms may account for someof the therapeutic effects of TCMs (Tang, Garg et al. 2009). Since allof these herbs and TCMs contain relatively high levels of nitrate andsome residual nitrite, there is in essence over 5000 years of phase Isafety trials in humans regarding nitrite and nitrate at these levels.

Human data: Based on the pre-clinical data obtained from above wecreated a rationally designed formulation based on the ingredients withthe greatest capacity for generating NO through the system in FIG. 8.Over two years of screening and testing a number of combinations ofdifferent herbs, we recognized a combination of beet root and hawthorneberry which gave us the most predictable and prolonged NO releaseprofile. The NO release kinetics are shown in FIG. 9A. In this in vitrosystem, the NO product has a half life of roughly 1 hour with respect toNO release.

The following formulation (Neo40) was prepared:

beet root powder, 200 mg;

hawthorn berry extract, 100 mg;

sodium nitrite, 20 mg;

L-citrulline, 100 mg;

vitamin B12, 1000 μg (as methylcobalamin and cyanocobalamin);

vitamin C, 100 mg (as magnesium ascorbate and ascorbic acid); and

about 250 mg to about 500 mg other ingredients (mannitol, modifiedcellulose, xylitol, natural orange flavor, magnesium stearate,sucralose, and silica).

Neo40 was sourced and developed into a quick dissolving lozenge by aGMP-certified facility. We then tested the pharmacokinetics of Neo40 inhumans. When allowed to dissolve in the mouth, Neo40 leads to a slow andsteady rise in plasma nitrite of humans as shown in FIG. 9B. Todemonstrate the difference between Neo40 and compositions of L-arginineand anti-oxidants that have been used as nutritional supplementsdesigned to enhance NO production, we compared Neo40 to one of the mostpopular products on the market. As shown in FIG. 9B, the L-arginineproduct did not lead to any appreciable formation of NO when consumed.This is in stark contrast to the NO formation resulting fromadministration of Neo40.

In order to demonstrate that Neo40 could restore NO levels, we conducteda small clinical trial in healthy volunteers (age 34-64; 6 male, 6female; 6 smokers, 6 non-smokers) and found that after taking Neo40 (onelozenge, twice a day) for 30 days lead to a modest but significantincrease in both plasma nitrite and nitrate (FIGS. 10A-B).

Based on this data, we began a clinical trial. Our inclusion criteriafor this double-blinded, placebo-controlled study were patients over 40with three or more of the following cardiovascular risk factors:hypertension, obesity, hyperlipidemia, smoking, sedentary lifestyle,family history of cardiovascular disease, and diabetes. As shown in FIG.10C, there was a modest to dramatic decease in triglycerides in thepatients with elevated triglycerides >149 mg/dL). As shown in FIG. 10D,100% of the patients with elevated triglycerides (>149 mg/dL) atbaseline had a reduction in their triglycerides with the twice-dailyregimen. Utilizing beet root and Hawthorne berry along with nitrite andL-citrulline provides a novel system for generating NO whereby themetabolism of nitrite is specifically directed towards reduction to NOby the Hawthorne berry. Though not to be bound by theory, we believe thepresence of an active reductase in the formulation is an importantaddition as it allows a more efficient means to produce NO from nitrite.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

What is claimed is:
 1. A composition, comprising: from about 40 weightparts to about 1000 weight parts of a botanical nitrate source; fromabout 20 weight parts to about 500 weight parts of a botanical source ofnitrite reduction activity; and from about 4 weight parts to about 100weight parts of a nitrite salt.
 2. The composition of claim 1, whereinthe botanical nitrate source is selected from the group consisting ofbeet root, artichoke, holy basil, gymnema sylvestre, L9H, ashwagandharoot, salvia, St. John wort, broccoli, stevia, spinach, gingko, kelp,tribulus, eleuthero, epimedium, eucommia, hawthorn berry, rhodiola,green tea, codonopsys, panax ginseng, astragalus, pine bark, dodderseed, Schisandra, cordyceps, and mixtures thereof.
 3. The composition ofclaim 1, wherein the botanical source of nitrite reduction activity isselected from the group consisting of hawthorn berry, Schisandra, greentea, beet root, pine bark, holy basil, gymnema sylvestre, L9H,ashwagandha root, salvia, St. John wort, broccoli, stevia, spinach,gingko, kelp, tribulus, eleuthero, epimedium, eucommia, rhodiola, greentea, codonopsys, panax ginseng, astragalus, dodder seed, cordyceps,berries, tea, beer, grapes, wine, olive oil, chocolate, cocoa, coffee,walnuts, peanuts, borojo, pomegranates, popcorn, yerba mate, andmixtures thereof.
 4. The composition of claim 1, wherein the nitritesalt is selected from the group consisting of sodium nitrite, potassiumnitrite, magnesium nitrite, calcium nitrite, and mixtures thereof. 5.The composition of claim 1, further comprising from about 20 weightparts to about 500 weight parts L-citrulline.
 6. The composition ofclaim 1, further comprising from about 0.2 weight parts to about 5weight parts vitamin B12.
 7. The composition of claim 6, wherein thevitamin B12 is in a form selected from the group consisting ofmethylcobalamin, cyanocobalamin, and mixtures thereof.
 8. Thecomposition of claim 1, further comprising from about 20 weight parts toabout 500 weight parts vitamin C.
 9. The composition of claim 8, whereinthe vitamin C is in a form selected from the group consisting ofmagnesium ascorbate, sodium ascorbate, potassium ascorbate, ascorbicacid, and mixtures thereof.
 10. The composition of claim 1, furthercomprising from about 50 weight parts to about 1500 weight parts of oneor more other ingredients selected from the group consisting ofmannitol, xylitol, sorbitol, other sugar alcohols, cellulose, celluloseesters, cellulose ethers, other modified celluloses, starch, modifiedstarches, other polysaccharides, oligosaccharides, disaccharides,saccharides, gelatin, polyvinylpyrrolidone, polyethylene glycol, otherbinders, flavorants, colorants, magnesium stearate, other antiadherentagents, other stearate salts, sweeteners, silica, and other lubricants.11. The composition of claim 10, wherein: the botanical nitrate sourceis selected from the group consisting of beet root, artichoke, holybasil, gingko, and mixtures thereof and is present at about 200 weightparts; the botanical source of nitrite reduction activity is selectedfrom the group consisting of hawthorn berry, Schisandra, green tea, beetroot, pine bark, and mixtures thereof and is present at about 100 weightparts; and the nitrite salt is selected from the group consisting ofsodium nitrite, potassium nitrite, and mixtures thereof and is presentat about 20 weight parts; and the composition further comprises: about100 weight parts L-citrulline; about 1 weight part vitamin B12 in a formselected from the group consisting of methylcobalamin, cyanocobalamin,and mixtures thereof; and about 100 weight parts vitamin C in a formselected from the group consisting of magnesium ascorbate, ascorbicacid, and mixtures thereof.
 12. The composition of claim 1, furthercomprising from about 20 weight parts to about 1000 weight partsL-arginine.
 13. The composition of claim 1, further comprising fromabout from about 20 weight parts to about 500 weight parts of a nitratesalt selected from the group consisting of sodium nitrate, potassiumnitrate, and mixtures thereof.
 14. The composition of claim 1, whereinthe composition is in a form of a lozenge dissolvable in the mouth. 15.The composition of claim 14, wherein the lozenge weighs from about 600mg to about 2000 mg.
 16. The composition of claim 11, wherein thecomposition is in a form of a lozenge dissolvable in the mouth.
 17. Thecomposition of claim 16, wherein the lozenge weighs from about 600 mg toabout 2000 mg.
 18. A method of reducing a patient's triglyceride level,comprising: administering to the patient a composition in a form of alozenge dissolvable in the mouth, the composition comprising: from about40 weight parts to about 1000 weight parts of a botanical nitratesource; from about 20 weight parts to about 500 weight parts of abotanical source of nitrite reduction activity; from about 20 weightparts to about 500 weight parts L-citrulline; and from about 4 weightparts to about 100 weight parts of a nitrite salt.
 19. The method ofclaim 18, wherein the botanical nitrate source is selected from thegroup consisting of beet root, artichoke, holy basil, gingko, andmixtures thereof and is present at about 200 weight parts; the botanicalsource of nitrite reduction activity is selected from the groupconsisting of hawthorn berry, Schisandra, green tea, beet root, pinebark, and mixtures thereof and is present at about 100 weight parts; theL-citrulline is present at about 100 weight parts; and the nitrite saltis selected from the group consisting of sodium nitrite, potassiumnitrite, and mixtures thereof and is present at about 20 weight parts;and the composition further comprises: about 1 weight part vitamin B12in a form selected from the group consisting of methylcobalamin,cyanocobalamin, and mixtures thereof; about 100 weight parts vitamin Cin a form selected from the group consisting of magnesium ascorbate,ascorbic acid, and mixtures thereof; and from about 50 weight parts toabout 1500 weight parts of one or more other ingredients selected fromthe group consisting of mannitol, xylitol, sorbitol, other sugaralcohols, cellulose, cellulose esters, cellulose ethers, starch, otherpolysaccharides, oligosaccharides, disaccharides, saccharides, gelatin,polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants,colorants, magnesium stearate, other antiadherent agents, other stearatesalts, sweeteners, silica, and other lubricants.
 20. The method of claim19, wherein the lozenge weighs from about 600 mg to about 2000 mg. 21.The method of claim 19, wherein the administering is performed once ortwice daily.
 22. A method of reducing a patient's C-reactive proteinlevel, comprising: administering to the patient a composition in a formof a lozenge dissolvable in the mouth, the composition comprising: fromabout 40 weight parts to about 1000 weight parts of a botanical nitratesource; from about 20 weight parts to about 500 weight parts of abotanical source of nitrite reduction activity; from about 20 weightparts to about 500 weight parts L-citrulline; and from about 4 weightparts to about 100 weight parts of a nitrite salt.
 23. The method ofclaim 22, wherein the botanical nitrate source is selected from thegroup consisting of beet root, artichoke, holy basil, gingko, andmixtures thereof and is present at about 200 weight parts; the botanicalsource of nitrite reduction activity is selected from the groupconsisting of hawthorn berry, Schisandra, green tea, beet root, pinebark, and mixtures thereof and is present at about 100 weight parts; theL-citrulline is present at about 100 weight parts; and the nitrite saltis selected from the group consisting of sodium nitrite, potassiumnitrite, and mixtures thereof and is present at about 20 weight parts;and the composition further comprises: about 1 weight part vitamin B12in a form selected from the group consisting of methylcobalamin,cyanocobalamin, and mixtures thereof; about 100 weight parts vitamin Cin a form selected from the group consisting of magnesium ascorbate,ascorbic acid, and mixtures thereof; and from about 50 weight parts toabout 1500 weight parts of one or more other ingredients selected fromthe group consisting of mannitol, xylitol, sorbitol, other sugaralcohols, cellulose, cellulose esters, cellulose ethers, starch, otherpolysaccharides, oligosaccharides, disaccharides, saccharides, gelatin,polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants,colorants, magnesium stearate, other antiadherent agents, other stearatesalts, sweeteners, silica, and other lubricants.